CN107949399A - For 10 antibody of anti-IL for the treatment of cancer and the combination product of CpG c-type oligonucleotides - Google Patents

Abstract

The present invention describes the therapeutic alliance comprising anti-10 antibody of IL or its antigen-binding fragment and CpG c-type oligonucleotides, and the therapeutic alliance is used for the purposes for the treatment of cancer.

Description

Combination for anti-IL-10 antibody and CpG-C the type oligonucleotides for the treatment of cancer Product

Invention field

The present invention relates to the therapeutic alliance available for treating cancer.In particular it relates to include anti-IL-10 antibody With the therapeutic alliance of TLR9 activators, the TLR9 activators are CpG-C type oligonucleotides.

Background of invention

The cytokine synthesis inhibitor factor or CSIF initially are referred to as, interleukin-10 (IL-10) is hematopoietic cell（Particularly Immunocyte）Effective immunomodulator.Th2 cells that cell such as activates, B cell, keratinocyte, monocyte and Macrophage produces IL-10.See, e.g., Moore et al.,Annu. Rev. Immunol. 11:165 (1993)。IL- 10 can suppress many cells（Including T cell, monocyte and macrophage）Activation and effector function.Specifically, IL-10 Suppress cell factor synthesis, including cell（Such as Th1 cells, natural killer cell, monocyte and macrophage）To IL-1, The synthesis of IFN-γ and TNF.See, e.g., Fiorentino et al.,J. Exp. Med., 170:2081-2095 (1989)；Fiorentino et al.,J. Immunol. 146:3444 (1991)；Hsu et al.,Int. Immunol. 4: 563 (1992)；Hsu et al.,Int. Immunol. 4:563 (1992)；D'Andrea et al.,J. Exp. Med. 178:1041 (1993)；De Waal Malefyt et al.,J. Exp. Med. 174:915 (1991)；Fiorentino etc. People,J. Immunol. 147:3815 (1991)。

Have confirmed tumor infiltrating macrophage, dendritic cells and CD4 in tumor microenvironment⁺And CD8⁺T cell pair The generation of IL-10 can suppress immune system tumor eradication (see, e.g., Jarnicki, et al. (2006)J. Immunol.896-904).Effective immunostimulatory activity and tumour root can be provided with the antagonist targeting IL-10 of IL-10 Remove.

The administration of some DNA sequence dnas (commonly referred to as immunostimulatory sequences) can induce be partial to the immune of Th1- types should Answer, as indicated by the secretion of the relevant cell factors of Th1-.Administration of the immunostimulating polynucleotides together with antigen can be led Cause the Th1- type immune responses for applied antigen.Roman et al. (1997) Nature Med. 3:849-854.Example Such as, the small of Escherichia coli (E.coli) beta galactosidase (β-Gal) in brine or adjuvant alum has been injected to intradermal Mouse by produce specific IgG1 and IgE antibody and secretion IL-4 and IL-5 but the CD4 of non-secretion of gamma-IFN⁺Cell And respond, so as to confirm that the T cell principally falls into Th2 subsets.But intradermal inject and (or drawn with tyne skins Hinder medicator（tyne skin scratch applicator）) encode β-Gal and the Plasmid DNA containing immunostimulatory sequences The mouse of (in brine) is by producing IgG2a antibody and secretion of gamma-IFN but does not secrete the CD4 of IL-4 and IL-5⁺Cell And respond, so as to confirm that the T cell principally falls into Th1 subsets.In addition, the specificity of the mouse of Plasmid DNA is injected IgE, which is produced, reduces 66-75%.Raz et al. (1996) Proc. Natl. Acad. Sci. USA 93:5141-5145.One As for, the response for naked DNA immunity inoculation is characterized in that the CD4 of antigenic stimulus⁺The IL-2 of T cell, TNF α and IFN-γ produces, this instruction Th1- type response.This is especially important in the treatment of allergia and asthma, such as the IgE of reduction Indicated by producing.The stimulation of immunostimulating polynucleotides has been confirmed with bacterial antigens, viral antigen and allergen The ability of Th1- type immune responses (see, e.g., WO 98/55495).There is a need in the art for the effect for improving cancer immunotherapy Power.

Summary of the invention

In one embodiment, the present invention provides a kind of method for being used to treat cancer in individual, the described method includes The therapeutic alliance comprising anti-IL-10 antibody and TLR9 activators is applied to the individual, wherein the TLR9 activators are CpG- C-type oligonucleotides.

In another embodiment, the present invention provides a kind of medicine for including anti-IL-10 antibody, its be used for TLR9 activators joint is used for treating cancer, wherein the TLR9 activators are CpG-C type oligonucleotides.In another embodiment party In case, the present invention provides a kind of medicine for including TLR9 activators, it is used for antibody combined for treating cancer with anti-IL-10 Disease, wherein the TLR9 activators are CpG-C type oligonucleotides.

Other embodiments provide purposes of the anti-IL-10 antibody in medicine preparation, and the medicine is when exciting with TLR9 For treating the cancer in individual, and purposes of the TLR9 activators in medicine preparation when agent is administered in combination, the medicine when with For treating the cancer in individual during the antibody combined administration of anti-IL-10.In such embodiments, the TLR9 activators It is CpG-C type oligonucleotides.

In another embodiment, the present invention provides anti-IL-10 antibody and TLR9 activators in medicine preparation Purposes, the medicine is used to treat the cancer in individual, wherein the TLR9 activators are CpG-C type oligonucleotides.Some In embodiment, the medicine includes kit, and the kit also includes package insert, and the package insert includes On the instruction of the cancer in anti-IL-10 Antybody therapies individual is used in combination with TLR9 activators.

Brief description

Fig. 1 shows the amino acid sequence of anti-IL-10 hum12G8, it is with SEQ ID NO:2 sequence of light chain and SEQ ID NO:1 sequence of heavy chain.CDR region domain is underlined.

Fig. 2 shows the amino acid sequence of anti-IL-10 TC40.11D8, it is with SEQ ID NO:3 light chain variable Region sequence and SEQ ID NO:4 weight chain variabl area sequence.

Fig. 3 shows the tumour growth of the injection tumour in the tumor model of mouse TC-1 both sides.Graph A shows each The volume of the injection tumour of animal and the number of every group of complete regression (CR).Chart B shows the middle position volume of injection tumour, Error bar indicates 68% confidential interval.Chart C daily compared for the injection gross tumor volume between treatment group.Chart D, which is shown, to be used for Contrast the unadjusted of the injection gross tumor volume between treatment and the P- values repeatedly adjusted.Unadjusted p value represents to be based on being directed to The bilateral p- values of the Peto ＆ Peto forms of the Gehan-Breslow non-parametric tests statistics of Right censored data.From being contrasted Two kinds treatment between 20,000 animals reallocate at random estimation P- values.P- values as the p- values expression repeatedly adjusted： Its through overregulate with control for given treatment between all time points by family's error rate.Adjusting be pass through by The maxT programs of Westfall and Young are applied to arranged distribution.

Fig. 4 shows the tumour growth of the non-injection tumour in the tumor model of mouse TC-1 both sides.Graph A is shown respectively The volume of the non-injection tumour of a animal and the number of every group of complete regression (CR).Chart B is shown in non-injection tumour Position volume, error bar indicate 68% confidential interval.Chart C daily compared for the non-injection gross tumor volume between treatment group.Chart D Show the P- values for contrasting the unadjusted of the non-injection gross tumor volume between treating and repeatedly adjusting.Unadjusted p value Represent the bilateral p- of the Peto ＆ Peto forms based on the Gehan-Breslow non-parametric tests statistics for Right censored data Value.Reallocate at random from 20,000 animals between the two kinds of treatments contrasted and estimate P- values.The p- value tables repeatedly adjusted Show such p- values：Its through overregulate with control for given treatment between all time points by family's error rate. Adjusting is by the way that the maxT programs of Westfall and Young are applied to arranged distribution.

The IFN α 2a in human PBMC's (2 donors) when Fig. 5 is shown with C59-08 and small control ODN 1040 processing 48 With the induction of IL-10.

Fig. 6 show with C59-08 processing 24 it is small when after IFN α in human renal cell carcinoma's tissue culture-can induce Gene (graph A), cell factor (chart B) and immune activation marker (chart C) mRNA expression induction.

Detailed description of the invention

Abridge through detailed description and embodiment of the invention, following abbreviation will be used：

The most preferably total responses of BOR

BID is each 2 times a day one

CBR clinical benefit rates

CDR complementarity-determining regions

CHO Chinese hamster ovaries

CR complete responses

DCR disease control rates

DFS is survived without disease

DLT dose limiting toxicities

The duration of DOR responses

Stable disease rate lasting DSDR

It is that FFPE formalin is fixed, paraffin embedding

FR framework regions

IgG immunoglobulin Gs

IHC immunohistochemistries are immunohistochemical

Related response standard is immunized in irRC

IV is intravenous

MTD maximum tolerated doses

NCBI American National Biotechnology Information centers

NCI national cancer institutes

ORR target response rates

OS is always survived

PD progressive diseases

PFS progresson free survivals

PR part responses

Q2W is one every 2 weeks

Q3W is one every 3 weeks

QD is one dose per day

Response evaluation criterions of the RECIST in solid tumor

SD stable diseases

VH immunoglobulin heavy chain variables area

VK immunoglobulin kappa light chains variable region.

I. define

In order to which the present invention can be more easily understood, some technical and scientific terms are defined particularly below.Unless herein Part other places especially define, and otherwise all other technical and scientific term used herein has of the art general The normally understood implication of logical technical staff.

As herein（Including the appended claims）It is middle to use, singulative such as "/kind " and " institute of word State " include their corresponding plural form, unless the context clearly indicates otherwise.

When applied to animal, people, subject, cell, tissue, organ or biofluid, " administration " represents external source medicine The contact with animal, people, main body, cell, tissue, organ or biofluid of thing, therapeutic agent, diagnosticum or composition.Term " is main Body " includes any organism, preferably animal, more preferably mammal (for example, rat, mouse, dog, cat, rabbit), and most preferably People.

Term " antibody " used herein shows desired bioactivity or combines any of the antibody of activity Form.Thus, it is used with most wide implication, and is specifically covered, but is not limited to, monoclonal antibody (including total length monoclonal Antibody), polyclonal antibody, multi-specificity antibody (for example, bispecific antibody), humanized antibody, human antibody, chimeric antibody With camelized single domain antibody." parental antibody " is in order to which desired use modifies antibody（Such as being treated as people The humanization of the antibody of agent）Immune system is exposed to antibody obtained from antigen before.

In general, basic antibody structural unit includes the tetramer.Each tetramer includes two identical polypeptide chains pair, Each to " light " chain (about 25 kDa) and " weight " chain (about 50-70 kDa).The amino terminal portion bag of every chain Include the variable region for being mainly responsible for about 100-110 of antigen recognizing an or more amino acid.The carboxy terminal half of heavy chain can limit Surely it is mainly responsible for the constant region of effector function.In general, people's light chain is categorized as κ and lambda light chain.In addition, usually by people's heavy chain point Class is μ, δ, γ, α or ε, and the isotype of antibody is respectively defined as IgM, IgD, IgG, IgA and IgE.In light chain and heavy chain Interior, variable region and constant region are connected by " J " region of about 12 or more amino acid, and wherein heavy chain further includes about 10 or more " D " region of multiple amino acid.Usually referring toFundamental Immunology(Paul, W., are compiled, second edition 7th chapter Raven Press, N.Y. (1989)。

The variable region of each light chain/heavy chain pair forms antibody combining site.Thus, it is however generally that, complete antibody has two A binding site.In addition in difunctional or bispecific antibody, described two binding sites are in general phase With.

In general, the variable domains of heavy chain and light chain include the three high change in relatively conservative framework region (FR) Area, also referred to as complementarity-determining region (CDR).The CDR aligns frequently by framework region, so as to be bound to defined epitope. In general, from N- ends to C- ends, light chain and heavy-chain variable domains all comprising FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.Distribution of the amino acid to each domain is typically according to defined below：Sequences of Proteins of Immunological Interest, Kabat, et al..; National Institutes of Health, Bethesda, Md.；5th edition；NIH publication numbers 91-3242 (1991)；Kabat (1978) Adv. Prot. Chem. 32: 1-75;Kabat, et al.,(1977) J. Biol. Chem. 252:6609-6616;Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883。

As used in this article, unless otherwise stated, " antibody fragment " or " antigen-binding fragment " represents antibody Antigen-binding fragment, that is, retain the antibody fragment for the ability for specifically combining the antigen combined by full length antibody, such as retains The fragment in one or more CDR region domains.The example of antibody binding fragment includes, but are not limited to Fab, Fab', F (ab')₂With Fv pieces Section；Binary；Straight chain antibody；Single-chain antibody molecules, for example, sc-Fv；Nano antibody（nanobodies）With by multiple antibody fragments The multi-specificity antibody of formation.

The antibody of " specifically combining " specific target protein is such antibody：Compared with other albumen, it shows excellent The target is first combined, but absolute binding specificity is not required in the specificity.If antibody binding can determine whether target protein in sample In presence, for example, not producing undesirable result such as false positive, then the antibody is considered being " special to its predetermined target The opposite sex ".Antibody for use in the present invention or its binding fragment by with than greatly at least 2 times of the affinity with non-target protein, preferably Greatly at least 10 times of ground, more preferably big at least 20 times and most preferably at least 100 times big affinity combination target protein.

Antibody as " chimeric antibody " expression：Wherein the part of heavy chain and/or light chain with from particular species (for example, People) or the corresponding sequence that belongs in the antibody of specific antibodies classification or subclass it is identical or homologous, while the remainder of the chain With from another species (for example, mouse) or belonging to another antibody isotype or the antibody of subclass and the fragment of this sample antibody In corresponding sequence it is identical or homologous, as long as they show desired bioactivity.

" human antibody " represents only to include the antibody of human immunoglobulin(HIg) protein sequence.If in mouse, in mouse cell In or in the hybridoma from mouse cell produce if, human antibody can contain mouse carbohydrate chain.Similarly, it is " small Mouse antibody " or " rat Ab " represent the antibody only comprising mouse or rat immunoglobulin sequence respectively.

" humanized antibody " is represented containing the antibody formation from inhuman (for example, mouse) antibody and the sequence of human antibody. Such antibody contains the minimum sequence from non-human immunoglobulin.In general, humanized antibody will include substantially institute At least one and usual two variable domains having, wherein all or essentially all of hypervariable loop is corresponding to inhuman The hypervariable loop of immunoglobulin, and all or essentially all of FR areas are the FR areas of human immunoglobulin sequence.People source Change at least one that antibody also optionally includes constant region for immunoglobulin (Fc) (being usually the constant region of human immunoglobulin(HIg)) Point.As necessary, prefix " hum ", " hu " or " h " is added to antibody cloning title, by humanized antibody and parent's grinding tooth Animal's antibody is distinguished and seen.The humanization form of rodent animal antibody generally comprises the identical CDR sequence of parent's rodent animal antibody, Although in order to increase affinity, increase the stability of humanized antibody or in order to which other reasons can be put including some amino acid Change.

Therapeutic scheme is used when referring to（All therapeutic alliances as described herein）During the cancer patient for the treatment of, " antitumor response " Refer at least one positive therapeutic effect, for example, the cancer cell count of reduction, the tumor size reduced, the cancer cell reduced Infiltrate into the speed in peripheral organs, the metastases reduced or tumor growth rate or progresson free survival.Can be with many sides Active treatment effect in formula measurement cancer is (referring to W. A. Weber, J. Null. Med. 50:1S-10S (2009)； Eisenhauer et al., ibid).In certain embodiments, using 1.1 standards of RECIST, two dimension irRC or one-dimensional IrRC, assesses the antitumor response to therapeutic alliance described herein.In certain embodiments, antitumor response be SD, PR, Any of CR, PFS or DFS.

" two-dimentional irRC " represented in Wolchok JD, et al. Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria.Clin Cancer Res. 2009;15(23):Standard set described in 7412-7420.These standards utilize the two of target focus Measurement of tumor is tieed up as a result, they by the longest diameter of each lesion by being multiplied by most long perpendicular diameter (cm²) and obtain.

" biopharmaceuticals " refer to a kind of biomolecule, such as antibody or fusion protein, it blocks any biological pathways In ligand/receptor signal transmission, such signal transmission support tumour maintain and/or growth or suppress antineoplastic immune should Answer.The classification of biopharmaceuticals including but not limited to for VEGF, EGFR, Her2/neu, other growth factor receptorses, CD20, The antibody of CD40, CD-40L, CTLA-4, OX-40,4-1BB and ICOS.

Usually with the cell growth of imbalance in term " cancer ", " carcinous " or " pernicious " expression or description mammal The physiological conditions being characterized.The example of cancer includes but is not limited to：Heart：Sarcoma (angiosarcoma, fibrosarcoma, band muscle Knurl, embryonal-cell lipoma), myxoma, rhabdomyosarcoma, fibroma, lipoma and teratoma；Lung：Bronchiogenic cancer (squamous cell, Undifferentiated cellule, undifferentiated maxicell, gland cancer), alveolar (bronchiole) cancer, bronchial adenoma, sarcoma, lymthoma, Cartilage hamartoma, celiothelioma；Stomach and intestine：Oesophagus (squamous cell carcinoma, gland cancer, leiomyosarcoma, lymthoma), stomach (cancer, lymthoma, Leiomyosarcoma), (duct adenocarcinoma, insulinoma, glucagonoma of pancreas, gastrinoma, carcinoid tumor, vasoactive are more for pancreas Peptide knurl), small intestine (gland cancer, lymthoma, carcinoid tumor, Kaposi sarcoma, liomyoma, hemangioma, lipoma, neurofibroma, fibre Tie up knurl), large intestine (gland cancer, tubular adenoma, villous adenoma, hamartoma, liomyoma) colorectum；Urogenital tract：Kidney (gland Cancer, wilm tumor [nephroblastoma], lymthoma, leukaemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, gland Cancer), prostate (gland cancer, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Cell plastid cancer, fibroma, adenofibroma, adenoma sample tumour, lipoma)；Liver：Hepatoma (hepatocellular carcinoma), cholangiocarcinoma cells, Hepatoblastoma, angiosarcoma, adenoma, hemangioma；Bone：Osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous Histocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulosarcoma), Huppert's disease, malignant giant born of the same parents Knurl chordoma, osteochondroma (osteocartilaginous exostosis), benign chondromas, chondrosarcoma, chondromyxoid fibroma, bone Osteoid osteoma and giant-cell tumor；Nervous system：Cranium (osteoma, hemangioma, granuloma, xanthoma, scleromalacia), meninx (meningioma, meningosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, Gonioma [pinealoma], glioblastoma multiforme, oligodendroglioma, schwann's cell tumor, retina are female thin Born of the same parents' knurl, congenital tumor), intraspinal cord neurinomas, meningioma, glioma, sarcoma)；Gynaecology：Uterus (endometrium Cancer), cervix (cervical dysplasias is bad before cervical carcinoma, tumour), ([slurries cystadenocarcinoma, mucinous cystadenocarcinoma, do not divide oophoroma ovary The cancer of class], granulosa theca cell tumour, Sai Ertuoli-Leydig cell tumour, dysgerminoma, malignant teratoma), vulva (squama Shape cell cancer, intraepithelial carcinoma, gland cancer, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubal (cancer), breast；Hematology：Blood (myeloid leukemia [acute and chronic], it is acute into Lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disease, Huppert's disease, myeloproliferative disorder Syndrome), Hodgkin's disease, non-Hodgkin lymphoma [malignant lymphoma]；Skin：Chromoma, basal-cell carcinoma, squamous are thin Born of the same parents' cancer, Kaposi sarcoma, mole dysplasia mole（moles dysplastic nevi）, lipoma, hemangioma, skin fiber Knurl, keloid, psoriasis；And adrenal gland：Neuroblastoma.In another embodiment, the cancer is cancer, lymph Knurl, leukaemia, blastoma and sarcoma.The more specifically example of such cancer includes：It is squamous cell carcinoma, myeloma, small thin Born of the same parents' lung cancer, non-small cell lung cancer, glioma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute myeloid leukaemia (AML), Huppert's disease, stomach and intestine (road) cancer, kidney, oophoroma, liver cancer, lymphoblastic leukemia, lymphocyte are white Blood disease, colorectal cancer, carcinoma of endometrium, kidney, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, Cancer of pancreas, glioblastoma multiforme, cervical carcinoma, the cancer of the brain, stomach cancer, carcinoma of urinary bladder, hepatoma, breast cancer, colon cancer and head Neck cancer.Another specific examples of cancer include clear-cell carcinoma.Another specific examples of cancer be non-Hodgkin lymphoma or The T- cell lymphomas of skin.Another specific examples of cancer are that acute myeloid leukaemia (AML) or myeloproliferative disorder are comprehensive Simulator sickness.

" CpG-C ODN " or " CpG-C types oligonucleotides " are the oligonucleotides of 12-100 bases longs, it has one A or multiple 5 '-TCG trinucleotides, wherein 5 '-T are apart from 5 '-end 0 of the oligonucleotides, 1,2 or 3 base, and at least The palindromic sequence of one at least eight bases longs includes one or more unmethylated CG dinucleotides.It is one or more A 5 '-TCG trinucleotides sequence can be separated with 5 '-end of the palindromic sequence by 0,1 or 2 base, or the palindrome Sequence can contain all or part of one or more of 5 '-TCG trinucleotide sequences.In one embodiment, institute It is oligodeoxynucleotide (ODN) to state oligonucleotides.In one embodiment, the oligonucleotides is 2 '-oligodeoxynucleotide. CpG-C ODN have stimulation B cell, inducing plasma cell sample dendritic cells (PDC) are ripe and cause high-caliber I types interferon The ability of the secretion of (for example, IFN-α, IFN-γ etc.).In certain embodiments, the CpG-C ODN are 12-100 alkali Base length, preferably 12-50 bases longs, preferably 12-40 bases longs, or preferably 12-30 bases longs. In some embodiments, the ODN be at least (lower limit) 12,13,14,15,16,17,18,19,20,21,22,23,24,25, 26th, 27,28,29,30,32,34,36,38,40,50,60,70,80 or 90 bases longs.In certain embodiments, it is described ODN be at most (upper limit) 100,90,80,70,60,50,49,48,47,46,45,44,43,42,41,40,39,38,37,36, 35th, 34,33,32,31 or 30 bases longs.In certain embodiments, at least one palindromic sequence is 8-97 alkali Base length, preferably 8-50 bases longs, or preferably 8-32 bases longs.In certain embodiments, it is described at least One palindromic sequence is at least (lower limit) 8,10,12,14,16,18,20,22,24,26,28 or 30 bases longs.Some In embodiment, at least one palindromic sequence be at most (upper limit) 50,48,46,44,42,40,38,36,34,32,30, 28th, 26,24,22,20,18,16,14,12 or 10 bases longs.In one embodiment, the oligonucleotides is few de- Oxygen nucleotide.In one embodiment, one or more of tnternucleotide linkage of the CpG-C ODN is modified Key.In one embodiment, one or more of tnternucleotide linkage of CpG-C ODN is thiophosphate (PS) key. In one embodiment, all tnternucleotide linkages of CpG-C ODN are thiophosphate (PS) keys.Phosphorothioate backbone represents All tnternucleotide linkages of CpG-C ODN are thiophosphate (PS) keys.

CpG-C type ODN and SEQ ID NO described herein:It is in their pharmaceutically acceptable salt shape that 13-26, which is, Formula, unless otherwise noted.Exemplary alkali salt includes：Ammonium salt, alkali metal salt, such as sodium, lithium and sylvite, alkali salt, example Such as calcium and magnesium salts, zinc salt, with organic base (for example, organic amine) such as N-Me-D- glucamines, N- [1- (2,3- dioleoyl oxygen Base) propyl group]-N, N, the salt that N- trimethyl ammonium chlorides, choline, tromethamine, dicyclohexyl amine, tert-butylamine are formed, and and amino The salt of the formation such as sour arginine, lysine.In one embodiment, it is in ammonium, sodium, lithium or potassium that the CpG-C types ODN, which is, Salt form.In a preferred embodiment, it is in sodium-salt form that the CpG-C types ODN, which is,.CpG-C ODN can be provided In the drug solution comprising pharmaceutically acceptable excipient.Alternatively, it is possible to CpG-C ODN are provided as lyophilized consolidate Body, then before administration reconstructs it in sterile water, brine or pharmaceutically acceptable buffer solution.

The present invention pharmaceutically acceptable excipient include for example, solvent, filler, buffer, tonicity contributor and Preservative (see, e.g., Pramanick et al., Pharma Times, 45:65-77, 2013).In some embodiments In, described pharmaceutical composition can be included and acted as the one or more in solvent, filler, buffer and tonicity contributor Excipient (for example, the sodium chloride in brine can serve as aqueous vehicles and tonicity contributor).The medicine group of the present invention Compound is suitable for parenteral administration.

In certain embodiments, described pharmaceutical composition includes aqueous vehicles as solvent.Suitable medium bag Include such as sterile water, saline solution, phosphate buffered saline (PBS) and Ringer's solution.In certain embodiments, the composition It is isotonic.

Described pharmaceutical composition can include filler.When described pharmaceutical composition will freeze before administration, filling Agent is particularly useful.In certain embodiments, the filler is in freezing or spray-drying process and/or is storing During aid in stabilizing active agent and prevent activating agent degrade protective agent.Suitable filler is sugar (monose, disaccharides and more Sugar) such as sucrose, lactose, trehalose, mannitol, sorbierite, glucose and gossypose.

Described pharmaceutical composition can include buffer.Buffer controls in processing, storage and optional restructuring procedure PH is with the degraded of inhibitory activity agent.Suitable buffer includes for example comprising acetate, citrate, phosphate or sulfate existing Interior salt.Other suitable buffers include such as amino acid such as arginine, glycine, histidine and lysine.It is described slow Electuary can also include hydrochloric acid or sodium hydroxide.In certain embodiments, the buffer maintains the pH of the composition In the range of 4-9.In certain embodiments, the pH is greater than (lower limit) 4,5,6,7 or 8.In some embodiments In, the pH is less than (upper limit) 9,8,7,6 or 5.That is, the pH is in the range of about 4-9, its lower limit is small In the upper limit.

Described pharmaceutical composition can include tonicity contributor.Suitable tonicity contributor include for example dextrose, glycerine, Sodium chloride, glycerine and mannitol.

Described pharmaceutical composition can include preservative.Suitable preservative includes such as antioxidant and antimicrobial Agent.But in preferred embodiments, described pharmaceutical composition aseptically prepares and is in disposable container In, and because without including preservative.

Term " palindromic sequence " or " palindrome is symmetrical " represent the nucleotide sequence of inverted repeat, for example, ABCDD ' C ' B ' A ', its Middle base（For example, A and A ', B and B ', C and C ', D and D '）Watson-Crick base-pair can be formed.Such sequence can be with It is single-stranded, or duplex structure can be formed, or hairpin ring structure can be formed under certain conditions.It is for example, used herein " the 8 base palindrome are symmetrical " represent as nucleotide sequence：Wherein palindromic sequence is 8 bases longs, such as ABCDD ' C ' B ' A’.Palindromic sequence can be the part of the also oligonucleotides containing non-palindromic sequence.Oligonucleotides can contain one or more Palindromic sequence part and one or more non-palindromic sequence parts.Alternatively, oligonucleotide sequence can be the complete palindrome. In the oligonucleotides with more than a palindromic sequence part, the palindromic sequence part can be with or without overlapping each other.

In one embodiment, CpG-C ODN of the invention are included：(a) 5’-N_x(TCG(N_q))_yN_w(X₁X₂CGX₂’ X₁’(CG)_p)_z,N_v(SEQ ID NO:13), wherein N is nucleosides, x=0,1,2 or 3, y=1,2,3 or 4, w=0,1 or 2, p =0 or 1, q=0,1 or 2, v=0-89 and z=1-20, X₁And X₁' it is from complementary nucleosides, X₂And X₂' it is from complementary core Glycosides, and wherein (TCG (N_q))_y5 ' end 0-3 bases of the 5 '-T of sequence apart from the oligonucleotides；At least eight alkali (b) The palindromic sequence of base length, wherein the palindromic sequence includes (X₁X₂CGX₂’X₁’(CG)_p)_zFirst (X of sequence₁X₂CGX₂’ X₁'), wherein the ODN is 12-100 bases longs.In certain embodiments, x=0, y=1, w=0, p=0 or 1, q=0,1 or 2, v=0-20 and z=1,2,3 or 4.In certain embodiments, X₁And X₂Individually A or T.In some implementations In scheme, the palindromic sequence has the base composition of the A and T more than 1/3.In certain embodiments, the CpG-C ODN Comprising selected from SEQ ID NO:The sequence of 16-26.

In certain embodiments, CpG-C ODN of the invention are by TCGN_q(X₁X₂CGX₂'X₁'CG)_zN_v (SEQ ID NO:14) form, wherein N is nucleosides, q=0,1,2,3,4 or 5, v=0-20, z=1-4, X₁And X₁' it is from complementary nucleosides, X₂ And X₂' it is certainly complementary nucleosides, and wherein described ODN is at least 12 bases longs.In certain embodiments, the CpG- C ODN are by selected from SEQ ID NO:The sequence composition of 16-26.

In certain embodiments, CpG-C ODN of the invention are by 5 '-TCGN_qTTCGAACGTTCGAACGTTN_s-3’ (SEQ ID NO:15) form, wherein N is nucleosides, q=0,1,2,3,4 or 5, s=0-20, and wherein described ODN is at least 12 bases longs.In one embodiment, s=0,1,2,3,4 or 5.In certain embodiments, the CpG-C ODN By being formed selected from following sequence：5’-TCGTTCGAACGTTCGAACGTTCGAA-3’(SEQ ID NO:17) q=0 and s = 4, 5’-TCGAACGTTCGAACGTTCGAACGTT-3’(SEQ ID NO:18) q=4 and s=0,5 '- TCGAACGTTCGAACGTTCGAACGTTCGAAT-3’(SEQ ID NO:20) q=4 and s=5,5 '- TCGTAACGTTCGAACGTTCGAACGTTA-3’(SEQ ID NO:21) q=5 and s=1, and 5 '- TCGTAACGTTCGAACGTTCGAACGTT-3’(SEQ ID NO:22) q=5 and s=0.

In one embodiment, the TLR9 activators be by sequence 5 '- TCGAACGTTCGAACGTTCGAACGTTCGAAT-3’(SEQ ID NO:20) the CpG-C ODN of composition.In another embodiment party In case, the CpG-C ODN are 5 ' TCGAACGTTCGAACGTTCGAACGTTCGAAT-3 ' (SEQ ID NO:20) sodium salt. In another embodiment, the CpG-C types oligonucleotides has by 5'-TCGTTCGAACGTTCGAACGTTCGAA-3' (SEQ ID NO:17) sequence of composition.In another embodiment, the CpG-C types oligonucleotides is 5'- TCGTTCGAACGTTCGAACGTTCGAA-3' (SEQ ID NO:17) sodium salt.

In another embodiment, the TLR9 activators CpG-C type oligonucleotides is selected from：

With

。

Table 1:The motif and sequence of CpG-C type oligonucleotides

。

It should be appreciated that for formula described herein or sequence motifs, any and all parameters are selected independently.For example, If x=0-2, y can be selected independently, no matter the value (or any other selectable parameter in formula) of x, as long as meeting total Oligonucleotide length limits.

The other CpG-C oligonucleotides of sequence included by motif with the present invention is suitable for disclosure used herein Method and medicine in.Multiple in addition CpG-C oligonucleotides descriptions of sequence included by motif with the present invention exist In the U.S. Patent number 7,745,606,8,158,768 and 8,871,732 of Dynavax Technologies Corporation. These sequences are incorporated by reference into hereby.

CpG ODN has been described in the art, and can readily determine that their work using standard test Property, the different aspect of the measure measurement immune response is (for example, the generation of cytokine secretion, antibody, NK cell activations, B cell Propagation, T cell propagation etc.).Exemplary method description is in the following documents：WO 97/28259; WO 98/16247; WO 99/11275, WO 98/55495 and WO 00/61151, and U.S. of Dynavax Technologies Corporation State's patent No. 7,745,606,8,158,768 and 8,871,732.Therefore, these and other method can be used for detecting and quantify The immunoregulatory activity of CpG ODN.

CpG-C oligonucleotides can contain modification.Suitable modification includes but is not limited to：3 ' OH or 5 ' OH groups are repaiied Decorations, the modification of nucleotide base, the modification of saccharic composition, and the modification of bound phosphate groups.It can include in palindromic sequence through repairing The base of decorations, as long as the modified base maintains the phase to its natural complement by Watson-Crick base pairing Homospecificity (for example, the palindromic portion of CpG-C oligonucleotides is kept from complementary).

CpG-C oligonucleotides can be straight chain, can be cricoid, or including annulus and/or hairpin loop.CpG- C oligonucleotides can be single-stranded or double-strand.CpG-C oligonucleotides can be DNA, RNA or DNA/RNA heterozygote.

CpG-C oligonucleotides can contain naturally occurring or modified non-naturally occurring base, and can contain Modified sugar, phosphate and/or end.For example, in addition to phosphodiester bond, phosphate modification includes but is not limited to：Phosphine Sour methyl esters, thiophosphate, phosphoramidate (bridging is non-bridged), phosphotriester and phosphorodithioate, and can with appoint Meaning is applied in combination.In certain embodiments, CpG-C oligonucleotides has only phosphorothioate bond, only phosphodiester bond or phosphorus The combination of acid diesters key and phosphorothioate bond.

As known in the art sugar-modified, such as 2 '-alkoxy-RNA analogs, 2 '-amino-RNA classes can also be made Like thing, 2 '-fluoro- DNA and 2 '-alkoxy-or amino-RNA/DNA chimeras and described herein other, and with any phosphate Modification combination.The example of base modification includes but is not limited to：Cytimidine from electrophilic part to CpG-C oligonucleotides (for example, 5- bromines cytimidine, 5- chlorine cytimidine, 5-flurocytosine, 5-iodocytosine) C-5 and/or C-6 and CpG-C oligonucleotides urine The addition of the C-5 and/or C-6 of pyrimidine (for example, 5-bromouracil, 5- chlorouracils, 5 FU 5 fluorouracil, 5-iodouracil).Such as Indicated above, application of the base modification in the palindromic sequence of CpG-C oligonucleotides should not interfere with Watson-Crick The self-complementarity of base involved in base pairing.But outside palindromic sequence, use is through repairing with can not having the limitation The base of decorations.For example, 2 '-O- methyl-uridines and 2 '-O- Methyl-Cytidines can be used outside palindromic sequence, however, it is possible to In inside and outside the use bromo- 2 '-deoxycytidines of 5- of palindromic sequence.Can be in other warps of the inside and outside use of palindromic sequence The nucleotide of modification includes 7- denitrogenation -8- azepines-dG, 2- amino-dA and 2- sulphur-dT.

The duplex (that is, double-strand) and hairpin formation of most of oligonucleotides are in dynamic equilibrium, wherein low widow's core GMP concentrations and higher temperature typically favor hair clip and are formed.Crosslinking can increase duplex or hair clip difference in covalent interchain or chain To heat-, ion-, pH- and concentration induction conformation change stability.Chemical crosslinking can be used to be locked in polynucleotides Duplex or hairpin formation are used for physical chemistry and biochemical characterization.The homogeneity and " locking " is in their most highly active shape in conformation The crosslinking oligonucleotides of formula (duplex or hairpin formation) may be more more active than their uncrosslinked homologue.Therefore, originally Some CpG-C oligonucleotides of invention contain crosslinking in covalent interchain and/or chain.

It is known in the art to be chemically crosslinked the various ways of duplex DNA.Any cross-linking method can be used, as long as handing over The polynucleotide products of connection have desired immunoregulatory activity.For example, a kind of method causes the end in duplex or hair clip Disulfide bond between the opposite thymidine of two of place.For the cross-linking method, with 5 '-DMT-N3- (tBu-SS- ethyls) thymidine -3 ' - Aminophosphite (" T* ") synthesizes target oligonucleotide.In order to form disulfide bond, by the disulfide bond reduction of mixing, by few nucleosides Acid purifying, hybridizes chain, and compound air oxidation is crosslinked to be formed in chain（In the case of hairpin formation）Or interchain is handed over Connection（In the case of duplex form）.Alternatively, it is possible to oligonucleotides is hybridized first, and then reduction, purifying and sky Gas aoxidizes.Describe in the art such method and other methods (see, e.g., Glick et al., J Org Chem, 56:6746-6747,1991, Glick et al., J Am Chem Soc, 114:5447-5448,1992, Goodwin etc. People, Tetrahedron Letters 35:1647-1650,1994, Wang et al., J Am Chem Soc, 117: 2981-2991,1995, Osborne et al., Bioorganic ＆ Medicinal Chemistry Letters, 6: 2339-2342,1996 and Osborne et al., J Am Chem Soc, 118:11993-12003, 1996).

Another cross-linking method is formed between the deviation residue (offset residues) in duplex or hairpin structure Disulfide bond.For the cross-linking method, target widow's core is synthesized with transformable nucleosides (commercially available from such as Glen Research) Thuja acid.This method utilizes, for example, A-A disulfide bond or C-A disulfide bond, and the connection for passing through other bases is also possible.For The polynucleotides of disulfide bond modification are formed, make the polynucleotides containing transformable nucleosides and cystamine (or other containing two sulphur The amine of key) reaction.In order to form disulfide bond, oligonucleotides is purified by the disulfide bond reduction of mixing, hybridizes chain, and will changed Compound air oxidation is crosslinked with being formed in chain（In the case of hairpin formation）Or interchain linkage（In the situation of duplex form Under）.Alternatively, it is possible to oligonucleotides is hybridized first, and then reduction, purifying and air oxidation.Describe in the art Such method (see, e.g., Ferentz et al., J Am Chem Soc, 113:4000-4002,1991, and Ferentz et al., J Am Chem Soc, 115:9006-9014, 1993).

The technology for being used to prepare polynucleotides and modification polynucleotides is known in the art.It is generally as follows synthesis and contains phosphorus The naturally occurring DNA or RNA of acid diesters key：Appropriate nucleosides aminophosphite is coupled to successively and is connected at 3 '-end 5 '-hydroxyl of oligonucleotides on solid support, in growth, is then oxidized to tricresyl phosphate by intermediate tris phosphite Ester.Once having synthesized desired polynucleotide sequence, lower polynucleotides just are removed from the holder, phosphotriester group is gone Di-phosphate ester is protected into, and by nucleoside base ammonium hydroxide or other alkali deprotection (see, e.g., Beaucage " Oligodeoxyribonucleotide Synthesis ", are shown in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, is compiled) Humana Press, Totowa, NJ, 1993;Warner et al., DNA 3:401,1984 and U.S. Patent number 4,458,066).

The oligonucleotides that CpG-C oligonucleotides can be modified containing phosphate, it is known that some of them can stablize the few core Thuja acid.Therefore, some embodiments include stabilized CpG-C oligonucleotides.Contain modified phosphoric acid ester bond or non-phosphoric acid The synthesis of the oligonucleotides of key is also known in the art (see, e.g., Matteucci " Oligonucleotide Analogs:An Overview ", are shown in Oligonucleotides as Therapeutic Agents, (D.J. Chadwick and G. Cardew, are compiled) John Wiley and Sons, New York, NY, 1997).Can be with few nucleosides The phosphorus derivant (or bound phosphate groups of modification) of sugar or the connection of sugar analogue part in acid can be phosplate, diphosphonic acid Ester, triguaiacyl phosphate, phosphonate ester, thiophosphate, phosphorodithioate, phosphoramidate etc..On being fully described The phosphate analogs that face is pointed out preparation and they nucleotide, modification nucleotide and oligonucleotides in incorporation in itself (see, e.g., Peyrottes et al., Nucleic Acids Res, 24:1841-1848, 1996;Chaturvedi etc. People, Nucleic Acids Res, 24:2318-2323, 1996；With Schultz et al., Nucleic Acids Res, 24:2966-2973, 1996).For example, in addition to oxidation step is replaced with vulcanisation step, phosphorothioate oligonucleotide Synthesis is similar to the synthesis (Zon " Oligonucleoside above for the description of naturally occurring oligonucleotides Phosphorothioates ", is shown in Protocols for Oligonucleotides and Analogs, Synthesis And Properties (Agrawal, compile) Humana Press, the 165-190 pages, 1993).

CpG-C oligonucleotides can include one or more ribonucleotides and (contain ribose as sugar solely or mainly Component), deoxyribonucleotide (containing deoxyribose as main saccharic composition), it is modified sugar or sugar analogue.Therefore, In addition to ribose and deoxyribose, sugar moieties can also be pentose, deoxypentose, hexose, deoxyhexamethylose, glucose, Arab Sugar, xylose, lyxose and sugar analogue cyclopenta.The sugar can be in pyranose or furyl glycosyl form.In CpG-C widow's core In thuja acid, the sugar moieties are preferably the furanoside of ribose, deoxyribose, arabinose or 2 ' -0- alkylriboses, and institute The various heterocyclic bases in anomeric configuration can be connected to by stating sugar.It is sugar-modified to include but is not limited to：2 '-alkoxy- RNA analogs, 2 '-amino-RNA analogs, 2 '-fluoro- DNA and 2 '-alkoxy-or amino-RNA/DNA chimeras.For example, Sugar-modified including but not limited to 2 '-O- methyl-uridines and 2 '-O- Methyl-Cytidines in CpG-C oligonucleotides.These sugar or sugar The preparation of analog and each kind of nucleosides (wherein such sugar or the like is connected to heterocyclic base (nucleic acid base)) is in itself It is known, therefore need not be described herein.Can also be made in the preparation of CpG-C oligonucleotides it is sugar-modified and with any phosphoric acid Ester modification combination.

Heterocyclic base or nucleic acid base in incorporation CpG-C oligonucleotides can be naturally occurring main purine and pyrimidine Base (i.e. uracil, thymidine, cytimidine, adenine and guanine, as described above), and the principal bases are natural Existing and synthesis modification.Thus, CpG-C oligonucleotides can include inosine, 2 '-BrdU and 2- amino -2 '-deoxidation One or more of adenosine.

" CBR " or " clinical benefit rate " refers to the SD of CR+PR+lasting.

" CDR " or " CDR " used herein refer to, unless otherwise noted, are defined using Kabat numbering systems immune Complementarity-determining region in globulin variable region.

" chemotherapeutant " is the chemical compound available for treating cancer.The species of chemotherapeutant includes but unlimited In：Alkylating agent, antimetabolite, kinase inhibitor, spindle poison plant alkaloid, cytotoxicity/antitumor antibiotic, It is topoisomerase enzyme inhibitor, photosensitizer, antiestrogenic agent and selective estrogen receptor modulators (SERM), anti-progesterone, female Adjusted (ERD) under hormone receptor, estrogen receptor antagon, luteinising hormone-releasing hormo activator, anti-androgens, fragrance The table of enzyme inhibitor, EGFR inhibitor, VEGF inhibitor and suppression in the gene involved in abnormal cell proliferation or tumour growth The antisense oligonucleotides reached.The chemotherapeutant for the treatment of method for use in the present invention is including cytostatic agent and/or carefully Cellular toxicity agent.

" Chothia " used herein refers in Al-LazikaniEt al., JMB273:927-948 is retouched in (1997) The antibody numbering system stated.

"comprising" or variation such as " comprising ", " containing " are used with inclusive implication in the present description and claims, That is, indicate the presence of the feature, but be not excluded for substantially strengthening operation or the effect of any embodiment of the present invention The presence or addition of further feature, unless since representation language or necessary hint context require in addition that.

" conservative modification variant " or " conservative substitution " is represented with having the similar characteristics (such as electric charge, side chain size, hydrophobic Property/hydrophily, Conformation of the main chain and rigidity etc.) other amino acid replacement albumen in amino acid so that the change can be through Often make, without the bioactivity for changing albumen or other desired characteristics, such as antigen affinity and/or specificity.Ability Known in field technique personnel, it is however generally that, the single amino acids displacement in the non-essential region of polypeptide will not substantially change life Thing activity is (see, e.g., Watson et al. (1987)Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., page 224 (the 4th edition)).In addition, in structure or functionally similar amino The displacement of acid is less likely to destroy bioactivity.Exemplary conservative substitution illustrates in the following Table 2.

The exemplary conservative amino acid replacement of table 2.

。

As used in the specification and claims, " substantially by ... form " and variation is such as " substantially By ... forming " instruction includes any key element enumerated or elements combination, and optionally includes with similar with the key element enumerated Or other key elements of different property, other key elements will not substantially change the dosage specified, method or composition Basic or new characteristic.As a non-limitative example, the anti-IL-10 being substantially made of the amino acid sequence enumerated Antibody can also include the one or more amino acid that will not substantially influence the characteristic of binding compounds, including one or more The displacement of amino acid residue.

" DCR " or " disease control rate " refers to CR+PR+SD.

" DSDR " or " lasting stable disease rate " refers to the SD of >=23 weeks.

" framework region " or " FR " used herein refer to the immune globulin variable region for not including CDR region domain.

" Kabat " used herein refers to by Elvin A. Kabat ((1991) Sequences of Proteins Of Immunological Interest, the 5th edition Public Health Service, National Institutes of Health, Bethesda, Md.) immunoglobulin advocated compares and numbering system.

" monoclonal antibody " used herein or " mAb " or " Mab " represent the colony of substantially homogeneous antibody, i.e. In addition to may be with a small amount of existing possible naturally occurring mutation, the antibody molecule of the colony be formed in amino acid sequence On be identical.On the contrary, conventional (polyclonal) antibody preparation generally includes many in their variable domains（Particularly they CDR）In have different aminoacids sequence different antibodies, they are often specific to different epitopes.Qualifier " Dan Ke Grand " the instruction antibody derives from the feature of substantially homologous antibody population, and should not be construed as needing by any spy Determine method and produce the antibody.For example, want monoclonal antibody used according to the invention can be by Kohler et al. (1975)Nature256:Prepared by 495 hybridoma methods described first, or can be by recombinant DNA method (see, e.g., the U.S. The patent No. 4,816,567) prepare.Also use in such as Clackson et al. (1991)Nature352:624-628 and Marks et al. (1991)J. Mol. Biol. 222:Technology described in 581-597 is " single from phage antibody library separation Clonal antibody ".Referring also to Presta (2005)J. Allergy Clin. Immunol. 116: 731。

When the specificity antineoplastic response for referring to the treatment to using therapeutic alliance described herein, " non-responder suffers from Person " refers to that the patient does not show antitumor response.

" ORR " or " target response rate " represents CR+PR, and ORR in certain embodiments_{(the 24th week)}The expression present invention Combined therapy 24 weeks after use the CR and PR measured in every patients of the irRECIST in group.

" patient " or " main body " represent to need to treat or participating in clinical test, epidemiological study or as control Any single main body, including the mankind and mammal veterinary science patient such as ox, horse, dog and cat.

" anti-IL-10 antibody " refers to combine IL-10 to suppress the active antagonist antibodies of IL-10.The replacement of IL-10 Title or synonym include：Interleukin-10, the cytokine synthesis inhibitor factor or CSIF.HIL-10 amino acid sequence can be with Referring to United States Patent (USP) 6217857.The amino acid sequence of ripe hIL-10 albumen is SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVM PQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYM TMKIRN (SEQ ID NO: 28)。

Useful anti-IL-10 antibody includes specificity in any for the treatment of method, medicine and purposes of the present invention The monoclonal antibody (mAb) of ground combination IL-10 or its antigen-binding fragment.The mAb can be human antibody, humanized antibody or Chimeric antibody, and human constant region can be included.In certain embodiments, the human constant region be selected from IgG1, IgG2, IgG3 and IgG4 constant regions, and in preferred embodiments, the human constant region is IgG1 or IgG4 constant regions.In some embodiments In, the antigen-binding fragment is selected from Fab, Fab '-SH, F (ab ')₂, scFv and Fv fragments.

In the certain preferred embodiments of the treatment method of the present invention, medicine and purposes, the anti-IL-10 antibody is Include the monoclonal antibody of region below or its antigen-binding fragment：(a) the SEQ ID NO of anti-IL-10 hum12G8: 5、6 With 7 light chain CDR and SEQ ID NO:8th, 9 and 10 heavy chain CDR.In the other of the treatment method of the present invention, medicine and purposes In preferred embodiment, the anti-IL-10 antibody is the monoclonal antibody or its antigen-binding fragment for including region below：(a) The SEQ ID NO of anti-IL-10 hum11D8:31st, 32 and 33 light chain CDR and SEQ ID NO:34th, 35 and 36 heavy chain CDR。

In other preferred embodiments of the treatment method of the present invention, medicine and purposes, the anti-IL-10 antibody is The monoclonal antibody or its antigen-binding fragment of hIL-10 are specifically combined, and contains SEQ ID NO comprising (a):11 or its The heavy chain variable region of variation, and (b) contain SEQ ID NO:The light chain variable region of 12 amino acid sequence or its variation.In this hair In the preferred embodiments other again of bright treatment method, medicine and purposes, the anti-IL-10 antibody is specifically to combine The monoclonal antibody of hIL-10 or its antigen-binding fragment, and contain SEQ ID NO comprising (a):4 or the heavy chain of its variation can Become area, and (b) contains SEQ ID NO:The light chain variable region of 3 amino acid sequence or its variation.Except with framework region At most 17 conservative amino acid replacements of (that is, in the outside of CDR) and preferably have in framework region be less than 10,9,8,7, Beyond 6 or 5 conservative amino acid replacements, the variation of weight chain variabl area sequence is identical with canonical sequence.Except with framework region In at most 5 conservative amino acid replacements (that is, in the outside of CDR) and preferably have and be less than 4,3 or 2 in framework region Beyond conservative amino acid replacement, the variation of light-chain variable sequence is identical with canonical sequence.

Table 3 below is provided for the exemplary anti-IL-10 in the treatment method of the present invention, medicine and purposes The list of the amino acid sequence of mAb, and the sequence is shown in Fig. 1-2.

" anti-IL-10 hum 12G8 variations " used herein refers to such monoclonal antibody：Except with place 3,2 or 1 conservative amino acid replacements at the position outside light chain CDR and 6,5,4,3,2 or 1 outside heavy chain CDR Beyond a conservative amino acid replacement（It is located at for example, becoming body position in FR areas or constant region）, it includes with anti-IL-10 hum Those identical heavy chains and sequence of light chain in 12G8.In other words, anti-IL-10 hum 12G8 and anti-IL-10 hum 12G8 variations include identical CDR sequence, but since the full-length light chains sequence and sequence of heavy chain respectively at them are no more than 3 It is different from each other with conservative amino acid replacement at a or 6 other positions.Anti- IL-10 hum 12G8 variations are with regard to following performance For it is substantially identical with anti-IL-10 hum 12G8：To the binding affinity of IL-10, and internal neutralizing effect.

" 1.1 response standards of RECIST " used herein refers in Eisenhauer et al., E.A. et al.,Eur. J. Cancer45:The definition illustrated in 228-247 (2009) on target focus or non-target focus, based on wherein when appropriate Measure the context of response.

When the specific antitumor response for referring to the treatment to using therapeutic alliance described herein, " respondent patient " is Refer to the patient for showing antitumor response.

" lasting response " refers to stop later longlasting treatments using the treatment of therapeutic agent or therapeutic alliance described herein Effect.In certain embodiments, the lasting response has the duration at least identical with the treatment duration, or at least It is treat the duration 1.5,2.0,2.5 or 3 times.

" histotomy " represents the single part or fragment of tissue sample, for example, the sample from normal structure or tumour is cut Under tissue slice.

" treatment " cancer used herein refers to, to cancer or being diagnosed to be the anti-IL-10 of administered of cancer and resist The therapeutic alliance of body and CpG-C type oligonucleotides, to realize at least one positive therapeutic effect, for example, the cancer cell of reduction Number, the tumor size of reduction, the cancer cell of reduction is infiltrated into the speed in peripheral organs, or the metastases or tumour reduced Growth rate.Can measure in many ways in cancer active treatment effect (referring to, W. A. Weber,J. Nucl. Med.50:1S-10S (2009)).For example, for Tumor growth inhibition, according to NCI standards, T/C≤42% is minimum anti-swollen Tumor activity is horizontal.T/C <10% is considered high anti-tumor activity level, the median tumor body of wherein T/C (%)=treated Median tumor volume × 100 of product/control.In certain embodiments, using 1.1 standards of RECIST or irRC (two dimension or One-dimensional) response of the assessment to therapeutic alliance described herein, and the treatment realized of the combination for passing through the present invention be PR, CR, OR, Any of PFS, DFS and OS.PFS, also referred to as the time of tumour progression " arrive ", instruction over the course for the treatment of with treatment with Cancer and has been subjected to including patient the amount of the time of CR or PR, and patient has passed through without the length of the time of growth afterwards Go through the amount of the time of SD.DFS represents to keep the length of the time without disease with patient after treatment over the course for the treatment of.OS is represented With originally or it is untreated individual or patient compared with life expectancy extension.In certain embodiments, to the combination of the present invention Response be any of PR, CR, PFS, DFS, OR and OS using 1.1 response criterion evaluations of RECIST.Effectively treat The therapeutic scheme of the combination of the invention of cancer patient can change with such as following factor：The morbid state of patient, age And weight, it is described to treat the ability for causing anticancer response in main body.An although embodiment of any aspect of the present invention Positive therapeutic effect can not possibly be effectively realized in every main body, but it will do in the main body of statistically significant number Arrive, as determined by by any statistical check known in the art, such as StudentShi t- inspections, Chi-square Test, basis The U- of Mann and Whitney is examined, Kruskal-Wallis examine (H- inspection), Jonckheere-Terpstra- inspections and Wilcoxon- is examined.

Term " therapeutic scheme ", " administration agreement " and " dosage regimen " are used interchangeably every in the combination to represent the present invention Plant the dosage of therapeutic agent and using opportunity.

" tumour " represents pernicious or potentially dislikes when it is applied to be diagnosed to be cancer or doubtful main body with cancer The tumour or tissue agglomerate of any size of property, and including primary tumor and secondary tumors.Solid tumor is the exception of tissue Growth or agglomerate, have been typically free of tumour or liquid regions.Different types of solid tumor with the type for forming their cell come Name.The example of solid tumor is sarcoma, cancer and lymthoma.Leukaemia（The cancer of blood）It is not generally formed solid tumor（State of the U.S. Vertical Cancer Institute（National Cancer Institute）, cancer glossary（Dictionary of Cancer Terms））.

" tumor load " is also referred to as " tumor burden ", represents to be dispersed throughout the total amount of internal anti-neoplastic.Tumor load Represent whole internal（Including lymph node and marrow）The sum of cancer cell or the overall size of tumour.By known in the art more Kind method, for example, the size by measuring tumour after being taken out from main body（For example, use caliper）, or make when in vivo With imaging technique, for example, ultrasound, bone scanning, computerized tomography (CT) or magnetic resonance imaging (MRI) scanning, can be true Determine tumor load.

Term " tumor size " represents that the total size of the tumour of the length and width of tumour can be measured as.Pass through this area Known a variety of methods, for example, the size by measuring tumour after being taken out from main body（For example, use caliper）, or work as Imaging technique is used when in vivo, for example, bone scanning, ultrasound, CT or MRI scan, it may be determined that tumor size.

" one-dimensional irRC " represented in Nishino M, Giobbie-Hurder A, Gargano M, Suda M, Ramaiya NH, Hodi FS. Developing a Common Language for Tumor Response to Immunotherapy: Immune-related Response Criteria using Unidimensional measurements. Clin Cancer Res.2013; 19(14):Standard set described in 3936-3943).These Standard utilizes the longest diameter (cm) of each lesion.

" variable region " used herein or " V areas " refers to the fragment of IgG chains, it is in sequence between different antibodies Variable.It extends to the Kabat residues 113 in Kabat residues 109 and heavy chain in light chain.

Any IL-10 antibody can be used in the combinations of the invention.In one embodiment, anti-IL-10 to be used Antibody is in US 8,226,947 and US 7,662,379（The disclosure of which is integrally incorporated by quoting hereby）Described in that A bit.In another embodiment, the anti-IL-10 antibody is anti-IL-10 hum12G8, and it includes two to have SEQ ID NO:The identical light chain of 2 sequence and two have SEQ ID NO:The identical heavy chain of 1 sequence.Contain coding hum12G8's The plasmid of the nucleic acid of heavy chain and light chain is deposited in ATCC on May 6th, 2004 respectively as PTA-5922 and PTA-5923.Another In one embodiment, the anti-IL-10 antibody is those described in U.S. Patent Publication No. US2012/0321617 (hVH20/hVL7, hVH20/hVL8, hVH26/hVL7 of humanization and chimeric cB-N10)).

II. method, purposes and medicine

In one aspect of the invention, the present invention provides a kind of method for being used to treat the cancer in individual, the method bag Include and apply the therapeutic alliance comprising anti-IL-10 antibody and CpG-C type oligonucleotides to the individual.

The therapeutic alliance can also include one or more other therapeutic agents.The other therapeutic agent can be, For example, chemotherapeutics, biopharmaceuticals, immunotherapeutic agent, immunogenicity medicament in addition to CpG-C type oligonucleotides（For example, The cancer cell of decrease, tumour antigen, the antigen presenting cell dendritic cells that antigen or nucleic acid stimulated such as derived from tumour, Immunostimulatory cells factor（For example, IL-2, IFN α 2, GM-CSF）With with encoding immune stimulating cell factor（Such as but not It is limited to GM-CSF）Gene transfection cell）And radiation.In certain embodiments, the immunotherapeutic agent include cell because One or more in son, small molecule adjuvants and antibody.In certain embodiments, the cell factor include chemotactic factor (CF), One or more in interferon, interleukin, lymphokine and tumor necrosis factor.The specific dosage of the other therapeutic agent It can be further change in dose plan, and optimal dose, drug dosage schedule and route of administration will specifically be controlled based on currently in use Treat agent and determine.

The example of chemotherapeutant includes：Alkylating agent such as phosphinothioylidynetrisaziridine and endoxan；Alkyl sulfonate esters such as busulfan, Improsulfan and piposulfan；Aziridines such as Benzodepa (benzodopa), carboquone, meturedepa (meturedopa) and uredepa (uredopa)；Aziridine and methyl melamine, including hemel, triethylene melamine, Triethylenephosphoramide, triethylene thiophosphoramide and trihydroxy methyl melamine；Annona lactone (acetogenin) is (especially It is bullatacin (bullatacin) and bullatacinone (bullatacinone))；Camptothecine (including the analog of synthesis is opened up Flutter and replace health)；Bryostatin; callystatin;CC-1065 (including its synthetic analogues Adozelesin, Ka Ze come it is new and Than Ze Laixin)；Cryptophycin (especially cryptophycin 1 and cryptophycin 8)；Dolastatin；Card meter Xing more (including Synthetic analogues KW-2189 and CBI-TMI)；Soft coral alcohol；Water ghost any of several broadleaf plants alkali；sarcodictyin；Spongistatin；Nitrogen mustards is all Such as Chlorambucil, Chlornaphazine, chlorine phosphamide (cholophosphamide), Estramustine, ifosfamide, mustargen, hydrochloric acid Nitromin, melphalan, novoembichin, phenesterin, prednimustine, Trofosfamide, uracil mustard；Nitro ureas such as blocks Mo Siting, chlorozotocin, Fotemustine, lomustine, Nimustine, Ranimustine；Antibiotics such as enediyne antibiotic (such as calicheamicin, especially calicheamicin γ 1I and calicheamicin phiI1, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)；Up to endomycin, including up to endomycin A；Diphosphonates, such as clodronate； Ai sibo mycin；And neoearcinostain chromophore and related chromoprotein enediyne antibiotic chromophore), aclacinomycin (aclacinomysin), D actinomycin D, anthramycin (authramycin), azaserine, bleomycin, act-C, Carabicin, carminomycin (caminomycin), cardinophyllin, chromomycin, dactinomycin D, daunorubicin, Detorubicin, 6- Diazo -5- oxn-l-norieucins, Doxorubicin (including morpholino-Doxorubicin, Cyanomorpholino-Doxorubicin, 2- Pyrrolin generation-Doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitogen it is mould Plain class such as mitomycin C, Mycophenolic Acid, nogalamycin, olivomycin, Peplomycin, porfiromycin (potfiromycin), Puromycin, triferricdoxorubicin, rodorubicin, broneomycin, streptozotocin, tubercidin, ubenimex, Zinostatin, assistant It is soft to compare star；Antimetabolite such as methotrexate (MTX) and 5 FU 5 fluorouracil (5-FU)；Folacin such as denopterin, first ammonia butterfly Purine, pteropterin, Trimetrexate；Purine analogue such as fludarabine, 6-MP, thiapurine, thioguanine；Pyrimidine is similar Thing such as ancitabine, azacitidine, 6- aza uridines, Carmofur, cytarabine, di-deoxyuridine, doxifluridine, according to promise His shore, floxuridine；Androgens such as Calusterone, dromostanolone propionate, epithioandrostanol, Mepitiostane, Testolactone；On anti-kidney Gland class such as aminoglutethimide, mitotane, Trilostane；Folic acid supplement such as folinic acid (frolinic acid)；In vinegar Portugal aldehyde Ester；Aldophosphamideglycoside；Aminolevulinic acid；Eniluracil；Amsacrine；bestrabucil；Bisantrene；Edatrexate (edatraxate)；Defosfamide (defofamine)；Demecolcine；Diaziquone；elformithine；Elliptinium Acetate；Ai Bo Mycin；Ethoglucid；Gallium nitrate；Hydroxycarbamide；Lentinan；Lonidamine；Maitansine class such as maitansine and ansamitocin；Rice Hold in the palm guanidine hydrazone；Mitoxantrone；Mopidamol；Nitracrine；Pentostatin；Phenamet；Pirarubicin；Losoxantrone；Podophyllic acid； 2- ethylhydrazides；Procarbazine；Razoxane；Rhizomycin；Sizofiran (sizofuran)；Spirogermanium；Acid is helped for slave；Triethyleneiminobenzoquinone； 2,2 ', 2 "-trichlorotriethylamine；Trichothecenes (especially T-2 toxin, verrucarine (verracurin) A, bar spore bacterium Plain A and snake rhzomorph)；Urethane (urethan)；Eldisine；Dacarbazine；Mannomustine；Dibromannitol；Dibromo is defended Lance alcohol；Pipobroman；gacytosine；Cytarabine (" Ara-C ")；Endoxan；Phosphinothioylidynetrisaziridine；Taxanes, such as Japanese yew Alcohol and Docetaxel；Chlorambucil；Gemcitabine；6- thioguanines；Mercaptopurine；Methotrexate (MTX)；Platinum analogs are such as suitable Platinum and carboplatin；Vincaleukoblastinum；Platinum；Etoposide (VP-16)；Ifosfamide；Mitoxantrone；Vincristine；Vinorelbine；Nuo Xiao Woods；Teniposide；Edatrexate；Daunomycin；Aminopterin；Xeloda；Ibandronate；CPT-11；Topoisomerase presses down Preparation RFS 2000；Difluoromethylornithine (DMFO)；Tretinoin such as retinoic acid；Capecitabine；With any of the above-described kind of medicine Acceptable salt, acid or derivative on.Further include it acts as the antihormone agent of the effect of adjusting or inhibitory hormone to tumour, Such as anti-estrogens and selective estrogen receptor modulators (SERM), including, for example, tamoxifen, Raloxifene, bend Lip river former times sweet smell, 4-hydroxytamoxifen, Trioxifene, Raloxifene, LY117018, Onapristone and Toremifene (Fareston)；Suppress the aromatase inhibitor of the aromatase enzyme of adjusting estrogen production in adrenal gland, for example, 4 (5)-imidazoles, Aminoglutethimide, megestrol acetate, Exemestane, formestane, Fadrozole, Vorozole, Letrozole and Anastrozole；Swash with anti-hero Plain class such as Flutamide, Nilutamide, Bicalutamide, Leuprorelin and Goserelin；With any of the above-described kind can pharmaceutically connect Salt, acid or the derivative received.

According to standard pharmaceutical practice, every kind of therapeutic agent in the therapeutic alliance of the present invention can be administered alone, or wrap Containing the therapeutic agent and one or more pharmaceutically acceptable carriers, excipient and diluent medicine (herein also by Referred to as pharmaceutical composition) in apply.

The present invention therapeutic alliance in every kind of therapeutic agent can simultaneously (that is, in identical medicine), concurrently (that is, in drug alone, being applied with any order a kind of later suitably using another kind) or in turn applied with any order With.When the therapeutic agent in the therapeutic alliance, in different dosage forms, (a kind of medicament is tablet or capsule and another medicament is sterile Liquid) and/or by the administration of different basis weights drug dosage schedule（For example, at least applying chemotherapeutics daily, biology is applied with lower frequency Therapeutic agent, such as 1 times a week, 1 time or 1 time every 3 weeks every 2 weeks）When, it is particularly useful to apply successively.

In certain embodiments, CpG-C type oligonucleotides is applied before anti-IL-10 antibody is applied, and in other realities Apply in scheme, CpG-C type oligonucleotides is applied after anti-IL-10 antibody is applied.In another embodiment, with it is anti- IL-10 antibody concurrently applies CpG-C type oligonucleotides.

In certain embodiments, intra-tumor or intravenously apply the CpG-C types oligonucleotides.In another reality Apply in scheme, intra-tumor or intravenously apply the anti-IL-10 antibody.In another embodiment, apply to intra-tumor With the CpG-C types oligonucleotides, and intravenously apply the anti-IL-10 antibody.

In certain embodiments, using when by least one of therapeutic alliance therapeutic agent be used as single therapy use Common same dose scheme (dosage, frequency and treatment duration) applies the medicament when identical cancer is treated.At it It is described compared with when at least one of therapeutic alliance therapeutic agent is used as single therapy in its common implementing scheme Patient receives the medicament of lower total amount, for example, the dosage of smaller, more low-frequency administration and/or shorter treatment continue Time.

Every kind of small molecule therapy agent in the therapeutic alliance of ground or the stomach and intestine other places administration present invention, including vein can be taken orally Interior, intramuscular, intraperitoneal, subcutaneous, rectum, part and transdermal administration approach.

The therapeutic alliance of the present invention can use before or after the surgical operation of tumour is removed, and can be controlled in radiation Before treatment, during or after use.

In certain embodiments, it is no before the therapeutic alliance of the present invention is administered to use biopharmaceuticals or Chemo-Therapy Treat the patient that agent was treated, i.e. be untreated.In other embodiments, the therapeutic alliance is administered to using life The patient of lasting response is not carried out after the prior treatment of thing therapeutic agent or chemotherapeutant, i.e. the patient is to live through Treatment.

The therapeutic alliance of the present invention is commonly used in tumour as treatment：Its is sufficiently large so that by palpation or passing through this The well-known imaging technique in field（Such as MRI, ultrasound or cat scan）It was found that.

Selection for the dosage (being also referred to as application program herein) of the therapeutic alliance of the present invention depends on Several factors, including the serum of entity or tissue turnover rate, the level of symptom, the immunogenicity of entity and individual being treated In target cell, the accessibility of tissue or organ.Preferably, dosage makes the amount for the every kind of therapeutic agent for being delivered to patient maximum Change, it is horizontal consistent with acceptable side effect.Therefore, the dosage of every kind of biopharmaceuticals in the combination and chemotherapeutant Amount and administration frequency are partly dependent on specific therapeutic agent, the order of severity of cancer being treated and patient characteristic.Selection is anti- The guidance of the suitable dosage of body, cell factor and small molecule is obtainable.See, e.g., Wawrzynczak (1996)Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;Kresina (eds.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY;Bach (eds.) (1993)Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY;Baert et al. (2003)New Engl. J. Med.348: 601-608;Milgrom et al. (1999)New Engl. J. Med. 341: 1966-1973; Slamon et al. (2001)New Engl. J. Med.344: 783-792;Beniaminovitz et al. (2000)New Engl. J. Med.342: 613-619;Ghosh et al. (2003)New Engl. J. Med. 348: 24-32; Lipsky et al. (2000)New Engl. J. Med. 343: 1594-1602; Physicians' Desk Reference 2003 (Physicians'Desk Reference, the 57th edition); Medical Economics Company; ISBN: 1563634457；57th edition (in November, 2002).Clinician can make determining for suitable dosage scheme, for example, using this Known in field or doubtful influence is treated or prediction can influence the parameter or factor for the treatment of, and depends on, for example, the clinic of patient History (for example, pervious treatment), the type of the cancer to be treated and stage and to one or more therapeutic agents in therapeutic alliance Response biomarker.

By continuous infusion, or by being periodically administered, for example, daily, it is 2 days every, 3 times a week or weekly, every 2 weeks, every 3 Week, monthly, every 2 months it is 1 inferior, the biopharmaceuticals in the therapeutic alliance of the present invention can be applied.Total weekly dosage is typically extremely Few 0.05 μ g/kg, 0.2 μ g/kg, 0.5 μ g/kg, 1 μ g/kg, 10 μ g/kg, 100 μ g/kg, 0.2 mg/kg, 1.0 mg/ Kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg weight or more.See, e.g., Yang et al. (2003)New Engl. J. Med. 349: 427-434;Herold et al. (2002)New Engl. J. Med. 346: 1692- 1698;Liu et al. people (1999)J.Neurol. Neurosurg. Psych.67: 451-456;Portielji et al. (2003) Cancer Immunol. Immunother. 52: 133-144。

In one embodiment of the invention, the anti-IL-10 antibody in the therapeutic alliance is anti-IL-10 hum 12G8, it intravenously is applied selected from following dosage：1 mg/kg Q3W、2 mg/kg Q3W、3 mg/kg Q3W、4 mg/ kg Q3W、5 mg/kg Q3W、6 mg/kg Q3W、7 mg/kg Q3W、8 mg/kg Q3W、9 mg/kg Q3W、10 mg/kg Q3W, 11 mg/kg Q3W, 12 mg/kg Q3W, 13 mg/kg Q3W, 14 mg/kg Q3W and 15 mg/kg Q3W.In this hair In another bright embodiment, the anti-IL-10 antibody in the therapeutic alliance is anti-IL-10 hum 12G8, it is 1 The dosage of mg/kg Q3W intravenously is applied.In another embodiment of the present invention, the anti-IL- in the therapeutic alliance 10 antibody are anti-IL-10 hum 12G8, its dosage in 3 mg/kg Q3W intravenously is applied.In another of the present invention In embodiment, the anti-IL-10 antibody in the therapeutic alliance is anti-IL-10 hum 12G8, it is 10 mg/kg Q3W's Dosage intravenously is applied.In other embodiments of the present invention, the anti-IL-10 antibody in the therapeutic alliance is anti- IL-10 hum 12G8, it intravenously applies in the dosage of 70 mg, 210 mg or 700 mg Q3W, optionally holds on day 1 Continue other 7 dosage.

In a preferred embodiment of the invention, the anti-IL-10 antibody in the therapeutic alliance is anti-IL-10 Hum 12G8 or anti-IL-10 hum 12G8 variations, it is applied selected from following dosage in liquid medicine：1 mg/kg Q3W、2 mg/kg Q3W、3 mg/kg Q3W、4 mg/kg Q3W、5 mg/kg Q3W、6 mg/kg Q3W、7 mg/kg Q3W、8 mg/kg Q3W、9 mg/kg Q3W、10 mg/kg Q3W、11 mg/kg Q3W、12 mg/kg Q3W、13 mg/kg Q3W、14 Mg/kg Q3W and 15 mg/kg Q3W.

In one embodiment of the invention, the CpG-C type oligonucleotides in the therapeutic alliance is SEQ ID NO: 20 oligonucleotides, it is applied selected from following dosage intra-tumor：0.1、0.5、1.0、2.0、3.0、4.0、5.0、6.0、 7.0th, 8.0 or 16.0 mg or 0.1-16 mg.In certain embodiments, 2 times a week, 1 times a week, every 2 weeks, 1 time every 3 weeks, Every month 1 time or every 2 months applies SEQ ID NO:20 CpG-C type oligonucleotides.In another embodiment of the invention In, SEQ ID NO are being applied selected from following dosage intra-tumor:20 CpG-C type oligonucleotides：Weekly 0.1,0.5,1.0, 2.0th, 3.0,4.0,5.0,6.0,7.0,8.0 or 16.0 mg or 0.1-16 mg, continue 4 times.In another implementation of the present invention In scheme, SEQ ID NO are being applied selected from following dosage intra-tumor:20 CpG-C type oligonucleotides：At the 1st and 8 day or 1st, 8,15 and 22 day, 0.1,0.5,1.0,2.0,3.0,4.0,5.0,6.0,7.0,8.0 or 16.0 mg.In the another of the present invention In one embodiment, SEQ ID NO are being applied selected from following dosage intra-tumor:20 CpG-C type oligonucleotides： 1st, 8,15,22,43,50,57 and 64 days, 2.0,4.0 or 8.0 mg.In other embodiments of the present invention, by SEQ ID NO:20 CpG-C types oligonucleotides was applied in the 1st, 8,15,22 day dosage intra-tumor in 1.0 or 4.0 mg, then Q3W, Optionally continue other 6 dosage.

CpG-C type oligonucleotides, its work with anti-IL-10 antibody can be applied according to any dosage and drug dosage schedule The dosage for effectively treating cancer with reaching together.Passed by the dosage escalation or dosage of one or both of these medicaments Subtract, it is possible to authenticate go out the optimal dose with the anti-IL-10 antibody of CpG-C type oligonucleotide combinatorials.In one embodiment, The therapeutic alliance includes the intravenous infusion of the anti-IL-10 hum 12G8 of 1 1-10 mg/kg and 1 times a week 1- every 3 weeks The SEQ ID NO of 16 mg:The intra-tumor of 20 CpG-C type oligonucleotides is applied.In another embodiment, the joint Treatment 2,4 or 8 mg comprising the intravenous infusion of the anti-IL-10 hum 12G8 of 1 time 1 or 3 mg/kg every 3 weeks and 1 times a week SEQ ID NO:The intra-tumor of 20 CpG-C type oligonucleotides is applied.In another embodiment, the therapeutic alliance includes The intravenous infusion of the anti-IL-10 hum 12G8 of 1 10 mg/kg and 1 times a week the SEQ ID NO of 2,4 or 8 mg every 3 weeks: The intra-tumor of 20 CpG-C type oligonucleotides is applied.In another embodiment, the therapeutic alliance is included every 3 weeks the 1st The intravenous infusion of the anti-IL-10 hum 12G8 of its 1,3 or 10 mg/kg and at the 1st, 8,15,22,43,50,57 and 64 day 4 The SEQ ID NO of mg:The intra-tumor of 20 CpG-C type oligonucleotides is applied.In another embodiment, the therapeutic alliance Include the intravenous infusion of the anti-IL-10 hum 12G8 of 1,3 or 10 mg/kg and weekly 4 mg on day 1 on day 1 every 3 weeks SEQ ID NO:The intra-tumor of 20 CpG-C type oligonucleotides apply it is for 4 weeks, then every 3 weeks.In another embodiment In, the therapeutic alliance include every 3 weeks on day 1 the intravenous infusion of the anti-IL-10 hum 12G8 of 1,3 or 10 mg/kg and The SEQ ID NO of 4 mg on day 1 weekly:The intra-tumor of 20 CpG-C type oligonucleotides apply it is for 4 weeks, be followed by 3 weeks It is disconnected, then weekly.In another embodiment, the therapeutic alliance includes the anti-of 1,3 or 10 mg/kg on day 1 every 3 weeks The intravenous infusion of IL-10 hum 12G8 continues at least 4 or 8 circulations, and at the 1st, 8,15,22 43,50,57 and 64 day 4 The SEQ ID NO of mg:The intra-tumor of 20 CpG-C type oligonucleotides is applied.In certain embodiments, the patient is joined Treatment treatment at least 12 weeks, 24 weeks is closed, for example, 8 circulate for 3- weeks.In other embodiments, by the patient with 10,11 or 12 doses of SEQ ID NO:20 CpG-C type oligonucleotide treatments.In certain embodiments, using the treatment of therapeutic alliance Continue until that the patient shows the sign of PD or CR.In a preferred embodiment, it is anti-in the therapeutic alliance IL-10 antibody is anti-IL-10 hum 12G8, by it on day 1 in 70 mg, 210 mg or the dosage vein of 700 mg Q3W Apply interiorly and continue other 7 dosage, and by SEQ ID NO:20 CpG-C types oligonucleotides is at the 1st, 8,15,22 day 1.0 Or 4.0 mg dosage intra-tumor apply, then Q3W applies other 6 dosage.In another preferred embodiment, institute It is anti-IL-10 hum 12G8 to state the anti-IL-10 antibody in therapeutic alliance, by it on day 1 in 210 mg or 700 mg Q3W Dosage intravenously apply, and by SEQ ID NO:20 CpG-C types oligonucleotides is at the 1st, 8,15,22 day 1.0 or 4.0 Apply to the dosage intra-tumor of mg, then Q3W.In a preferred aspect of embodiments above, when with CpG-C type oligonucleotides When applying anti-IL-10 hum 12G8 on the same day, first using CpG-C type oligonucleotides.One in embodiments above is excellent In terms of choosing, SEQ ID NO:20 CpG-C type oligonucleotides is oligodeoxynucleotide and has phosphorothioate backbone.In the above Another preferred aspect of embodiment, CpG-C type oligonucleotides are SEQ ID NO:20 sodium salt, the SEQ ID NO:20 It is the oligodeoxynucleotide with phosphorothioate backbone.

Present invention provides the medicine for including anti-IL-10 antibody as described above and pharmaceutically acceptable excipient. When the anti-IL-10 antibody is biopharmaceuticals（For example, mAb）When, can using regular growth culture and recycling/purification technique To produce the antibody in Chinese hamster ovary celI.The anti-IL-10 antibody can be freezed in buffer solution and reconstructed for vein Interior injection.Present invention provides the medicine comprising TLR9 activators and pharmaceutically acceptable excipient, wherein the TLR9 Activator is CpG-C type oligonucleotides.CpG-C types oligonucleotides can be reconstructed in physiology buffer solution and be noted for intra-tumor Penetrate.

Medicine described herein can be used as kit to provide, and the kit includes the first container and second container and bag Fill specification.The first container contains at least one medicine for including anti-IL-10 antibody, and the second container contains at least One medicine for including CpG-C type oligonucleotides, and the package insert or label are included on using the drug therapy The guidance of the cancer of patient.The first container and second container can be by same or different shapes (for example, bottle, note Emitter and bottle) and/or material (for example, plastics or glass) composition.The kit is further included in medicament administration can The useful other materials of energy, such as diluent, filter, IV bags and pipeline, syringe needle and syringe.

In some embodiments of the above treatment method of the present invention, medicine and purposes, the individual is people, and described Cancer is solid tumor, and in certain embodiments, the solid tumor is carcinoma of urinary bladder, breast cancer, clear cell renal carcinoma, head and neck Squamous cell carcinoma, squamous cell lung carcinoma, chromoma, non-small cell lung cancer (NSCLC), oophoroma, cancer of pancreas, prostate Cancer, clear-cell carcinoma (RCC), Small Cell Lung Cancer (SCLC) or triple negative breast cancer.In certain embodiments, the cancer It is the squamous cell carcinoma or melanoma of NSCLC, carcinoma of endometrium, urothelium cancer, head and neck.

In other embodiments of the above treatment method of the present invention, medicine and purposes, the individual is people, and described Cancer is ferroheme malignant tumour, and in certain embodiments, the ferroheme malignant tumour is Acute Lymphoblastic Leukaemia (ALL), acute myeloid leukaemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Diffusivity large B cell lymthoma (DLBCL), the DLBCL of the EBV- positives, the big B- cell lymphomas of Primary Mediastinal, rich in T- The big B- cell lymphomas of cell/tissue cell, follicular lymphoma, Hodgkin lymphoma (HL), lymphoma mantle cell (MCL), Huppert's disease (MM), -1 albumen of myeloid cell leukaemia (Mcl-1), myelodysplastic syndrome (MDS), skin T- are thin Born of the same parents' lymthoma, non-Hodgkin lymphoma (NHL) or small lymphocytic lymphoma (SLL).

In an embodiment of above treatment method, medicine and purposes, the individual is people, and the cancer is selected from black Plain knurl, neck squamous cell carcinoma, breast cancer and non-Hodgkin lymphoma.In another embodiment, the cancer is metastatic Or unresectable melanoma, late period neck squamous cell carcinoma, the breast cancer of corium transfer or painless non-Hodgkin lymphoma. In another embodiment, the patient have anti-PD1 treat fail metastatic or unresectable melanoma, Late period neck squamous cell carcinoma, the breast cancer of corium transfer, at least one prior treatment being in progress after radiation have lost The painless non-Hodgkin lymphoma lost.In another embodiment, the cancer be selected from melanoma, head and neck cancer, breast cancer and B- cell lymphomas.

In an embodiment of above treatment method, medicine and purposes, the individual is people, and the cancer is selected from kidney Cell cancer, non-small cell lung cancer, carcinoma of urinary bladder and colorectal cancer.

From the teaching contained by this paper these and other aspects of the invention can be made apparent（Including the example being listed below Property specific embodiment）.

The particular exemplary embodiments of the present invention

1. a kind of method for being used to treat the cancer in individual, the described method includes apply to include anti-IL-10 to the individual The therapeutic alliance of antibody or its antigen-binding fragment and TLR9 activators, wherein the TLR9 activators are CpG-C type few nucleosides Acid.

2. the method for embodiment 1, wherein the anti-IL-10 antibody is monoclonal antibody.

3. a kind of medicine comprising anti-IL-10 antibody or its antigen-binding fragment, it is used to combine with TLR9 activators For treating the cancer in individual, wherein the anti-IL-10 antibody is monoclonal antibody or its antigen-binding fragment, and it is described TLR9 activators are CpG-C type oligonucleotides.

4. a kind of medicine for including TLR9 activators, it is used to combine with anti-IL-10 antibody or its antigen-binding fragment For treating the cancer in individual, wherein the TLR9 activators are CpG-C type oligonucleotides.

5. the medicine of embodiment 3 or 4, the medicine also includes pharmaceutically acceptable excipient.

6. the purposes of anti-IL-10 antibody or its antigen-binding fragment in medicine preparation, the medicine is worked as to swash with TLR9 For treating the cancer in individual when dynamic agent is administered in combination, wherein the TLR9 activators are CpG-C type oligonucleotides.

7. the purposes of TLR9 activators in medicine preparation, the medicine is worked as and anti-IL-10 antibody or its antigen binding For treating the cancer in individual when fragment is administered in combination, wherein the TLR9 activators are CpG-C type oligonucleotides.

8. the purposes of anti-IL-10 antibody or its antigen-binding fragment and TLR9 activators in medicine preparation, the medicine Thing is used to treat the cancer in individual, wherein the TLR9 activators are CpG-C type oligonucleotides.

A kind of 9. kit for including the first container, second container and package insert, wherein the first container includes At least one medicine containing anti-IL-10 antibody or its antigen-binding fragment, the second container include it is at least one containing The medicine of TLR9 activators, and the package insert includes the guidance on the cancer using the drug-treated individual, its Described in TLR9 activators be CpG-C type oligonucleotides.

10. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body or its antigen-binding fragment include SEQ ID NO:11 and SEQ ID NO:12 heavy chain and light chain variable region.

11. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body or its antigen-binding fragment include：(a) SEQ ID NO:5th, 6 and 7 light chain CDR and SEQ ID NO:8th, 9 and 10 weight Chain CDR.

12. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body is the anti-IL-10 monoclonal antibodies comprising heavy chain and light chain, and wherein described heavy chain includes SEQ ID NO:1, and it is described light Chain includes SEQ ID NO:2.

13. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body is anti-IL-10 hum 12G8 or anti-IL-10 hum 12G8 variations.

14. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body or its antigen-binding fragment include：(a) SEQ ID NO:31st, 32 and 33 light chain CDR and SEQ ID NO:34th, 35 and 36 heavy chain CDR.

15. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body is the anti-IL-10 monoclonal antibodies comprising heavy chain and light chain, and wherein described heavy chain includes SEQ ID NO:29, and it is described Light chain includes SEQ ID NO:30.

16. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein the anti-IL-10 resists Body is the anti-IL-10 monoclonal antibodies comprising heavy chain and light chain variable region, and wherein described heavy chain variable region includes SEQ ID NO:4, and the light chain variable region includes SEQ ID NO:3.

17. the method for any one of embodiment 1-16, medicine, purposes or kit, wherein the CpG-C types are few Nucleotide is made of following sequence：(a) 5’-N_x(TCG(N_q))_yN_w(X₁X₂CGX₂’X₁’(CG)_p)_z,N_v(SEQ ID NO:13), Wherein N be nucleosides, x=0,1,2 or 3, y=1,2,3 or 4, w=0,1 or 2, p=0 or 1, q=0,1 or 2, v=0-89 and Z=1-20, X₁And X₁' it is from complementary nucleosides, and X₂And X₂' it is from complementary nucleosides；At least eight bases longs (b) Palindromic sequence, wherein the palindromic sequence includes (X₁X₂CGX₂’X₁’(CG)_p)_zFirst (X of sequence₁X₂CGX₂’X₁'), wherein institute It is 12-100 bases longs to state oligonucleotides.

18. the method for embodiment 17, medicine, purposes or kit, x=0, y=1, w=0, p=0 or 1, q= 0th, 1 or 2, v=0-20 and z=1,2,3 or 4.

19. the method for any one of embodiment 1-16, medicine, purposes or kit, wherein the CpG-C types are few Nucleotide is by TCGN_q(X₁X₂CGX₂'X₁'CG)_zN_v(SEQ ID NO:14) form, wherein N is nucleosides, q=0,1,2,3,4 or 5, v=0-20, z=1-4, X₁And X₁' it is from complementary nucleosides, X₂And X₂' it is from complementary nucleosides, and wherein described oligonucleotides It is at least 12 bases longs.

20. the method for any one of embodiment 1-16, medicine, purposes or kit, wherein the CpG-C types are few Nucleotide is by 5 '-TCGN_qTTCGAACGTTCGAACGTTN_s-3’(SEQ ID NO:15) form, wherein N is nucleosides, q=0,1, 2nd, 3,4 or 5, s=0-20, and wherein described oligonucleotides is at least 12 bases longs.

21. the method for any one of embodiment 1-16, medicine, purposes or kit, wherein the CpG-C types are few Nucleotide is selected from：

With

。

22. the method for any one of embodiment 1-16, medicine, purposes or kit, wherein the CpG-C types are few Nucleotide have byComposition Sequence.

23. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein CpG-C types widow's core Thuja acid have byThe sequence of composition.

24. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein CpG-C types widow's core Thuja acid is SEQ ID NO:The sodium salt of 17 sequence, and the oligonucleotides is the few deoxyribonucleoside for having phosphorothioate backbone Acid.

25. the method for any one of embodiment 1-9, medicine, purposes or kit, wherein CpG-C types widow's core Thuja acid is SEQ ID NO:The sodium salt of 17 sequence, and the oligonucleotides is the few deoxyribonucleoside for having phosphorothioate backbone Acid.

26. a kind of method for being used to treat the individual human for being diagnosed to be cancer, the described method includes to described individual weekly Apply SEQ ID NO to the dosage intra-tumor of 1-16 mg:20 CpG-C types oligonucleotides and with 1 1-10 mg/kg every 3 weeks Dosage intravenously apply anti-IL-10 hum 12G8, preferably with the dosage intra-tumor of the mg of weekly 1,2,4,8 or 16 Using SEQ ID NO:20 CpG-C types oligonucleotides and intravenously applied with the dosage of 11,3 or 10 mg/kg every 3 weeks Anti- IL-10 hum 12G8.

27. a kind of medicine for including anti-IL-10 hum 12G8, it is used for and SEQ ID NO:20 CpG-C type widow's cores Thuja acid is used in combination for treating the cancer in individual human, wherein by SEQ ID NO:20 CpG-C type oligonucleotides is weekly The individual is administered to the dosage intra-tumor of 1-16 mg, and by anti-IL-10 hum 12G8 in 1 1-10 mg/kg every 3 weeks Dosage intravenously apply, preferably wherein by SEQ ID NO:20 CpG-C types oligonucleotides is in weekly 1,2,4,8 or 16 The dosage intra-tumor of mg it is administered to the individual, and by anti-IL-10 hum 12G8 11,3 or 10 mg/kg's every 3 weeks Dosage intravenously is applied.

28. a kind of medicine for including anti-IL-10 hum 12G8, it is used for and SEQ ID NO:20 CpG-C type widow's cores Thuja acid is used in combination for treating the cancer in individual human, wherein by SEQ ID NO:20 CpG-C type oligonucleotides is weekly It is for 4 weeks to be administered to the dosage intra-tumor of 1-16 mg the individual, then every 3 weeks 1 time, and by anti-IL-10 hum 12G8 Intravenously applied in the dosage of 1 1-10 mg/kg every 3 weeks.

29. a kind of medicine for including anti-IL-10 hum 12G8, it is used for and SEQ ID NO:20 CpG-C type widow's cores Thuja acid is used in combination for treating the cancer in individual human, wherein by the anti-IL-10 hum 12G8 on day 1 70 mg, The dosage of 210 mg or 700 mg Q3W intravenously are applied, and by SEQ ID NO:20 CpG-C types oligonucleotides the 1st, 8th, apply to 15,22 days dosage intra-tumors in 1.0 or 4.0 mg, then Q3W.

30. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is entity Knurl.

31. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is bladder Cancer, breast cancer, clear cell renal carcinoma, head/neck squamous cell carcinoma, squamous cell lung carcinoma, chromoma, non-small cell lung cancer (NSCLC), oophoroma, cancer of pancreas, prostate cancer, clear-cell carcinoma, Small Cell Lung Cancer (SCLC) or triple negative breast cancer.

32. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is NSCLC, RCC, carcinoma of endometrium, urothelium cancer, the squamous cell carcinoma or melanoma of head and neck.

33. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is acute Lymphoblastic leukemia (ALL), acute myeloid leukaemia (AML), chronic lymphocytic leukemia (CLL), chronic Myelogenous Leukaemia (CML), diffusivity large B cell lymthoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), jacket cell leaching Bar knurl (MCL), Huppert's disease (MM), -1 albumen of myeloid cell leukaemia (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), cutaneous T-cell lymphomas or small lymphocytic lymphoma (SLL).

34. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is melanocyte Knurl, the squamous cell carcinoma of head and neck, breast cancer or B- cell lymphomas.

35. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is transfer Property or unresectable melanoma, late period neck squamous cell carcinoma, the breast cancer of corium transfer or painless non-Hodgkin lymphoma.

36. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is anti- PD1 or anti-CTLA-4 treat the metastatic or unresectable melanoma to fail, the evening being in progress after radiation The painless non-Hodgkin lymphoma that phase neck squamous cell carcinoma, the breast cancer of corium transfer, at least one prior treatment have failed.

37. the method for any one of embodiment 1-29, medicine, purposes or kit, wherein the cancer is selected from kidney Cell cancer, non-small cell lung cancer, carcinoma of urinary bladder and colorectal cancer.

38. method, medicine, purposes or the kit of any one of embodiment 1-16 and 26-37, wherein described CpG-C type oligonucleotides is SEQ ID NO:The sodium salt of 20 sequence, and the oligonucleotides is with phosphorothioate backbone Oligodeoxynucleotide.

39. method, medicine, purposes or the kit of any one of embodiment 1-16 and 26-37, wherein described CpG-C type oligonucleotides is SEQ ID NO:20 sequence, and the oligonucleotides is that the widow with phosphorothioate backbone takes off Oxygen nucleotide.

Conventional method

In molecular biology standard method description Sambrook, Fritsch and Maniatis (1982 and 1989 second editions, 2001 the 3rd editions)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001)Molecular Cloning, the 3rd edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, volume 217, Academic Press, San Diego, CA).Standard method also appears in Ausbel, et al. (2001)Current Protocols in Molecular Biology, the 1-4 volumes, John Wiley and Sons, Inc. New York, NY, which depict in bacterial cell Clone and DNA mutagenesis (volume 1), clone's (volume 2), glycoconjugate and protein expression in mammalian cell and yeast (volume 3) and bioinformatics (volume 4).

Describe the method for protein purification, including immune precipitation, chromatography, electrophoresis, centrifugation and crystallization (Coligan, Et al. (2000)Current Protocols in Protein Science, volume 1, John Wiley and Sons, Inc., New York).Describe chemical analysis, chemical modification, posttranslational modification, the production of fusion protein, the glycosyl of albumen Change (referring to,For example,Coligan, et al. (2000)Current Protocols in Protein Science, volume 2, John Wiley and Sons, Inc., New York;Ausubel, et al. (2001)Current Protocols in Molecular Biology, volume 3, John Wiley and Sons, Inc., NY, NY, the 16.0.5- 16.22.17 page; Sigma-Aldrich, Co. (2001)Products for Life Science Research, St. Louis, MO；The 45-89 pages; Amersham Pharmacia Biotech (2001)BioDirectory, 384-391 pages of Piscataway, N.J., the).Describe polyclonal and the production of monoclonal antibody, purifying and fragmentation (Coligan, et al. (2001)Current Protcols in Immunology, volume 1, John Wiley and Sons, Inc., New York;Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;Harlow and Lane, ibid).For Characterization ligand/receptor interaction standard technique be it is available (referring to,For example,Coligan, et al. (2001)Current Protocols in Immunology, volume 4, John Wiley, Inc., New York)。

Can prepare monoclonal antibody, polyclonal antibody and humanized antibody (referring to,For example,Sheperd and Dean (eds.) (2000)Monoclonal Antibodies, Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001)Antibody Engineering, Springer-Verlag, New York;Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor 139-243 pages of Laboratory Press, Cold Spring Harbor, NY, the;Carpenter, et al. (2000)J. Immunol. 165:6205;He, et al. (1998)J. Immunol. 160:1029;Tang et al. (1999) J. Biol. Chem. 274:27371-27378;Baca et al. (1997)J. Biol. Chem. 272:10678-10684; Chothia et al. (1989)Nature342:877-883;Foote and Winter (1992)J. Mol. Biol. 224: 487-499；U.S. Patent number 6,329,511).

One alternative solution of humanization is used in the human antibody library that is shown on bacteriophage or in transgenic mice Human antibody library (Vaughan et al. (1996)Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine1:837-839;Mendez et al. (1997)Nature Genetics 15:146-156; Hoogenboom and Chames (2000)Immunol. Today21:371-377;Barbas et al. (2001)Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York;Kay et al. (1996)Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, CA;De Bruin et al. (1999) Nature Biotechnol. 17:397-399)。

Necessary to the purifying of antigen and non-antibody produce.The cellular immunity animal with target antigen can be used.Then Can be from immunized animal separating Morr. cell, and the splenocyte can be made to be merged with myeloma cell line to produce hybridoma (referring to,For example,Meyaard et al. (1997)Immunity7:283-290;Wright et al. (2000)Immunity 13: 233-242; PrestonEt al., ibid;Kaithamana et al. (1999)J. Immunol. 163:5157- 5164)。

Antibody and for example small drug molecule, enzyme, liposome, polyethylene glycol (PEG) can be made conjugated.Antibody can be used for controlling Treat, diagnosis, kit or other purposes, and including with such as dyestuff, radio isotope, enzyme or metal (for example, colloidal gold) The antibody of coupling is (see, e.g., Le Doussal et al. (1991)J. Immunol. 146:169-175; Gibellini Et al. (1998)J. Immunol. 160:3891-3898;Hsing and Bishop (1999)J. Immunol. 162: 2804-2811;Everts et al. (2002)J. Immunol. 168:883-889)。

For the method for flow cytometry, including fluorescence activated cell sorts (FACS), be it is available (referring to,For example,Owens, et al. (1994)Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, the 2nd Version; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ).It is suitable for modification of nucleic acids（Including the nucleic acid primer as such as diagnostic reagent With probe, polypeptide and antibody）Fluorometric reagent be available (Molecular Probesy (2003)Catalogue, Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO)。

Describe immune system histological standard method (referring to,For example,Muller-Harmelink (eds.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY;Hiatt, et al. (2000)Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA;Louis, et al. (2002)Basic Histology: Text and Atlas, McGraw-Hill, New York, NY)。

For determining such as antigen fragment, targeting sequencing, protein folding, functional domain, glycosylation site and sequence ratio To software kit and database be it is available (referring to,For example,GenBank, Vector NTI®Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher®(TimeLogic Corp., Crystal Bay, Nevada);Menne, et al. (2000)Bioinformatics16: 741-742;Menne, et al. (2000)Bioinformatics Applications Note16:741-742;Wren, et al. (2002)Comput. Methods Programs Biomed. 68:177- 181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690)。

Embodiment

Embodiment 1：Immunological regulations of the C59-08 to people's cell

C59-08 is that have phosphorothioate backbone and the oligodeoxynucleotide of 5 ' OH and 3 ' OHSodium salt.

Use standard Ficoll separation methods Ficoll-Paque^TM PLUS (GE Healthcare Bio- Sciences, Pittsburgh, PA) from the buffy coat from two donors separate human peripheral blood mononuclear cell (PBMCs).By separated PBMC in the phosphate-buffered containing 2% hyclone (FBS) and 2 mM ethylenediamine tetra-acetic acids (EDTA) Washing 2 times in brine (PBS).By the cell settling flux and blue or green containing 10%FBS, 2 mM L-Glutamines, 100 U/mL With 1 × 10 in the RPMI 1640 of mycin and 100 μ g/mL streptomysins⁶A cells/well is cultivated in the U- base plates of 96- holes.By institute Cell is stated in the presence of C59-08 or 7 μM of control ODN 1040 for having 0.016 μM of dosage to 5 μM of scopes in constant humidity culture With the final volume of 0.2 mL in 37 DEG C, 5%CO in case₂It is lower culture 48 it is small when.Supernatant is harvested and uses Meso Scale Discovery people's IFN α 2a and hIL-10 Tissue culture reagents box (Rockville, MD) measure IFN α 2a and IL-10.

The results show is in Figure 5.IFN α 2a and IL-10 in C59-08 induction human PBMCs are produced, and optium concentration is in 0.2 μ M。

Embodiment 2:Immunological regulations of the C59-08 to human tumour sample

Human tumour tissue culture

It is big from commercial source (Bio-Options, Folio, Coversant Bio and Boston BioSource) and Rochester Learn（University of Rochester）Obtain the human tumour sample from patient.

It is interior when after surgical operation 1 is small to collect fresh tumor tissue and be placed on AQIX transport mediums (AQIX, UK) In.4 DEG C of transports overnight will be organized in Merck Research Laboratories, Palo Alto, CA.

The tumour is embedded in UltraPure^TMIn low melting-point agarose (Invitrogen, Carlsbad, CA) simultaneously Use Mcllwain^TMTissue Chopper (Stoelting Co., Wood Dale, IL) and into 400 μm.First will be swollen Knurl section is fixed on Millicell-CM cell culture inserts (Millipore, Billerica, CA), and is trained in constant humidity Support in case in 37 DEG C, 5%CO₂Under in air and supplemented with 4.5 g/L glucose, L-Glutamine, Sodium Pyruvate (Mediatech, Inc., Manassas, VA)、10%FBS (SAFC Biosciences, Lenexa, Kansas)、100 The interface culture of 1 ml DMEM culture mediums of U/ml penicillin and 100 ug/ml streptomysins.

By tumor biopsy when culture 24 is small in the presence of having 0.1,0.5 and 1 μM of C59-08 or 1 μM of control ODN 1040. By tumor sample in dry ice snap frozen and before treatment 37 DEG C preservation.

RNA is separated and real-time quantitative PCR

Using polytron homogenizers by total serum IgE by homogenization separate into RNA STAT-60 (Tel-Test, Friendswood, TX) in.Total serum IgE is extracted according to the scheme of manufacturer.After isopropanol precipitating, total serum IgE is used and is mutually locked Light test tube phenol:Chloroform:Isoamyl alcohol (25:24:1) (Sigma-Aldrich, St. Louis, MO) is extracted again.

According to the scheme of manufacturer using QuantiTect Reverse Transcription (Qiagen, Valencia, CA), the processed total serum IgE of reverse transcription DNA enzymatic.From Life Technologies (Foster City, CA) Commercially available primer.On Fluidigm Biomark sequence detection systems (Fluidigm, Foster City, CA) Perform using the unlabelled primer of respective 900 nM and 250 nM the FAM- probe marked in TAQMAN RTqPCR reactions From the real-time quantitative PCR of 10 ng cDNA of each sample.The level of ubiquitin is measured in one individually reaction, and for passing through Δ-Δ Ct methods are by data normalization.Using the ubiquitin of each sample and average period threshold value (Ct) value of target gene, use The equation below obtains normalized value：1.8^{(Ct ubiquitin-Ct target genes)}×10⁴。

Handling result

It is straight in clear-cell carcinoma (RC) (n=5), non-small cell lung cancer (NSCLC) (n=3) and carcinoma of urinary bladder (n=1) and colon In intestinal cancer (n=1) tissue culture with C59-08 induction IFN α-derivable gene (IFN α 2, MCP1, MCP2, OAS2, IP-10, GBP1, ISG-54, MxB and TRAIL), cell factor (IFN β, IL-10, IL-12, IL-6 and TNF α) and immune activation Marker (CD80, CD86, CD40, CD70 and OX40L) carries out the ex vivo treatment of human tumour.Use the sample from RCC donors Representative data show in figure 6：(A) IFN α-derivable gene；(B) cell factor；(C) immune activation mark Thing.

Embodiment 3：Antitumor activity of the combination of anti-IL-10 and intra-tumor C59-08 in animal model

TC40.11D8 is the 1/ κ monoclonal antibodies of mouse IgG for targeting mouse IL-10.1 isotype controls of mouse IgG are to adenopathy Malicious 5 specific mouse monoclonal antibody of h exon 2s.Two kinds of antibody all derive from internal next as freezing (- 80 DEG C) reserve Source.

The preparation of antibody

Formulation Buffer is that every kind of antibody specificity albumen and is prevented from precipitating to stablize.TC40.11D8 and mouse IgG 1 are same The preparation of kind of type control is 75 mM sodium chloride, 10 mM sodium phosphates, 3% sucrose, pH7.3.

Oligodeoxynucleotide

Phosphorothioate oligodeoxynucleotides (ODN) CpG 1826 based on cytidine-phosphate-guanosine (CpG)(InvivoGen, San Diego, CA) is that mouse TLR 9 is special The activator of property.CpG 1826 has CpG class Type B sequences.Based on CpG thiophosphate ODN C59-08 (Dynavax, Berkeley, CA) it is the activator for activating people and mouse TLR 9.C59-08 has CpG class c-type sequences.Compare ODN (Dynavax, Berkeley, CA) With the non-CpG sequences containing phosphorothioate backbone.

The preparation of oligodeoxynucleotide

CpG 1826 is reconstructed in the concentration of 2 mg/mL in 0.9% sodium chloride, decile, and in -20 DEG C of preservations.C59-08 is existed The reconstruct in phosphate buffered saline (PBS) (PBS) of the concentration of 4.53 mg/mL, decile, and in -20 DEG C of preservations.Control ODN is existed The concentration of 4.47 mg/mL reconstructs in PBS, decile, and in -20 DEG C of preservations.

Animal

About 7-8 week old female C57BL/6J mouse derive from Jackson Laboratory (Sacramento, CA).Arbitrarily obtain Take regular animal food and water.Before the study starts by animal stable breeding 1 week.The animal when research starts (i.e. tumour is implanted into) Average weight is 19 grams.

It is related to the nursing of animal and the program used in research and passes through Merck research laboratories（Merck Research Laboratories）Zoopery management group（Institutional Animal Care and Use Committee） Evaluation and approval.In the course of the research, according in Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)、Animal Welfare Act、American Veterinary Medical Association (AVMA) Euthanasia Panel on Euthanasia and Institute for Laboratory Animal Research (ILAR) Guide to the Care and Use of Laboratory Principle described in the guide of Animals, carries out the nursing and use of animal.

Tumor cell line prepares and implantation

The TC-1 cell lines provided by Johns Hopkins University (Baltimore, MD) are derived from and use human papilloma Mouse primary pulmonary epithelial cells (Lin KY et al. of virus (HPV-16) E6 and E7 and c-Ha.ras oncogene cotransformationsCancer Res.1996 Jan 1, 56(1):21-6).TC-1 cells are isogenic with C57BL6/J strains.

TC-1 cells are cultivated in the DMEM supplemented with 10% hyclone and 0.4 mg/mL Geneticins.By subconfluent Hypodermically (SC) is injected into (1 × 10 in the waist flank of every animal TC-1 cells in 0.1 mL serum-frees DMEM⁵In right flank In abdomen and 0.5 × 10⁵In left flank abdomen).First by animal in the electric shears shaving of the region for implantation.

Measurement of tumor and weight

In first dose the previous day and hereafter 2 times a week, tumour is measured.Length of tumor and width are measured using electronic caliper, And determine gross tumor volume using the following formula：Volume (mm³)=0.5 × length x width², wherein length is longer dimension. First dose the previous day and hereafter 2 times a week, animal is weighed.Bias in order to prevent, removes any of weight or gross tumor volume Outlier, and remaining mouse is divided into each treatment group based on the gross tumor volume (tumour for being referred to as injection) in right flank abdomen.

Prepared to drug solns

The freezing reserve of antibody is melted and is transferred to wet ice., will be per bottle reserve in order to avoid the freezing repeated is melted Melt once, and aliquot is prepared in the volume of enough first uses.By the low adhesion test tube of polypropylene for this purpose.Will Aliquot is in -80 DEG C of preservations.Before each administration, an aliquot is melted and is diluted in appropriate diluent Nominal concentration.Before each administration, by the aliquot of ODN (control ODN, CpG 1826 and C59-08) melt and Nominal concentration is diluted in 0.9% sodium chloride.

The administration of antibody and oligodeoxynucleotide

At the 0th, 4,8 and 12 day in 10 mg/kg peritonaeums (IP) apply isotype controls mIgG1 and anti-IL-10 mIgG1. (IT) control ODN (2.5 mg/kg), CpG 1826 (1 were applied to intra-tumor only in right tumour at the 0th, 4,8 and 12 day ) and C59-08 (2.5 mg/kg) mg/kg.

Statistical method

Gross tumor volume is contrasted before treatment daily in follow-up.The follow-up of each animal can because of excessive tumor burden or its Its reason more early terminates.Tumor size depending on the reason and when last time measures, last time is observed Gross tumor volume processing is for the animal all with posteriori volume lower bound (Right censored data).

In order to contrast Liang Ge treatment groups in given day, the non-parametric Mann-Whitney for allowing Right censored data is used (or Wilcoxon orders summation) is examined extensive：Peto the and Peto forms that Gehan-Breslow is examined.From two contrasted 20,000 animals between secondary treatment are reallocated at random estimates bilateral p- values.In order to control given treatment in institute's having time Point presses family's error rate, by the way that the maxT programs of Westfall and Young repeatedly are adjusted p- values applied to arranged distribution. Statistical significance is defined using the p- values less than 0.05.

In order to describe purpose, daily and treatment group volume is summarized by their median.In order to allow to check, right Pass through Kaplan-Meier methods using GreenwoodShi formula in number scale（With confidence belt）Estimate daily and treatment group Distribution function.Median is estimated as the 50% of distribution function, there is the confidential interval obtained by inverting confidence belt.Use 68% Confidence level, by with for summary data it is common " it is in the form of average value ± SE " suitable because the latter is about the 68% of average value Confidential interval.

When the follow-up of animal more early terminates, the data of animal are handled causality classification and as follows：(1) tumor load： Right censorship during last time measured value；(2) tumor ulceration is formed:The right censorship in last time measured value, it is assumed that this exceedes threshold It is worth (1000 mm³)；Otherwise the time afterwards ignores animal；(3) weight saving/sick (including finds dead, has sick Sign):Time afterwards ignores animal；(4) unrelated with treatment (for example, chance on death, without sick sign, Management terminates):The right censorship in last time measured value, it is assumed that this exceedes threshold value (1000 mm³)；Otherwise the time afterwards Ignore animal.

Treatment results

When the average external volume of the tumour in right flank abdomen reaches about 60 mm³ (39 mm³ - 87 mm³) when, before first dose The C57BL/6J mouse for carrying TC-1 tumours were divided into 5 treatment groups in one day：(1) mIgG1 isotype controls+control ODN； (2) mIgG1 isotype controls+C59-08；(3) anti-IL-10+CpG 1826；(4) anti-IL-10+control ODN；With (5) anti-IL-10+C59-08.It is 0 mm in the scope of the gross tumor volume in left side³ - 113 mm³.Complete by tumour disappears (CR) it is defined as, it is assumed that animal packet that day is being measurable by tumour, does not have measurable tumour when measuring.

The results show is in figures 3 and 4.Combined with intra-tumor CpG 1826 (the 3rd group) or C59-08 (the 5th group) anti- IL-10 causes the CR (Fig. 3 A) of the injection tumour at least 3 animals.But the anti-IL-10 (only combined with C59-08 5 groups) cause the CR (3 in 10 animals) (Fig. 4 A) of non-injection tumour.Including C59-08 single therapies (the 2nd group) Other treatments without result in injection or non-injection tumour CR.

With randomized controlled treatment, anti-IL-10 single therapies are compared with C59-08 single therapies, are combined with C59-08 (IT) The administration of anti-IL-10 causes the 6th, the 9 and 12 day injection gross tumor volume (p being substantially reduced<0.05, between time point repeatedly Adjust) (Fig. 3 B-D).Compared with randomized controlled treatment and anti-IL-10 single therapies, with C59-08 (IT) the anti-IL-10's combined Using causing the 6th, the 9 and 12 day non-injection gross tumor volume (p being substantially reduced<0.05, repeatedly adjusted between time point) (figure 4B-D)。

Embodiment 4:The anti-IL-10 hum 12G8 combined with C59-08 in the main body with late tumor

1b phase dose escalation studies are carried out, it tests the HUM12G8 of the ascending-dose combined with the dosage level of C59-08. The research uses the amplification group proposed by using 3+3 designs of standard in the MTD or MAD of HUM12G8.With late period The main body of tumour（Tumour, which is presented on, injects reachable position）It is qualified for research.It is qualified that there is late tumor Main body will include：The metastatic or unresectable melanoma that anti-PD1 treatments have failed, has been in progress after radiation Late period head and neck squamous cell carcinoma, corium transfer breast cancer, at least one prior treatment fail it is painless it is non-suddenly Strange gold lymthoma or B- cell lymphomas.All main bodys in each group should be that TLR activators or anti-IL10 are not treated 's.

4 test of cure of table

。

Dosage escalation

Following three administration groups are selected to increase and ascending-dose together with administration with the C59-08 of higher fixed dosage The immune activation of HUM12G8 combinations.All dosage levels in the A of part will comply with 3+3 designs.Continue the dosage of HUM12G8 It is incremented by differentiate preliminary MTD or MAD.The administration of each group is as follows：

1) 1 mg C59-08 intra-tumors and 70 mg HUM12G8 intravenously

2) 4 mg C59-08 intra-tumors and 70 mg HUM12G8 intravenously

3) 4 mg C59-08 intra-tumors and 210 mg HUM12G8 intravenously

4) 4 mg C59-08 intra-tumors and 700 mg HUM12G8 intravenously.

If 4 mg C59-08 and 70 mg HUM12G8 combinations (group 2) are not resistant to, then are later group (3 Hes 4) C59-08 is decremented to 1 mg dosage.

All references cited herein is all incorporated by reference into, such as every single publication, database bar Mesh (such as Genbank sequences or GeneID entries), patent application or patent are specifically and individually pointed out by quoting simultaneously Enter.U. S. application 62/169,321 and 62/168,470 is incorporated herein by reference.Applicant is according to 37 C.F.R. § 1.57 (b) (1) be intended to the statement that is incorporated by reference into this represent each and every single publication, data base entries (such as Genbank sequences or GeneID entries), patent application or patent, each of which is according to 37 C.F.R. § 1.57 (b) (2) clearly differentiate, even if such reference is not next to the special statement being incorporated by reference into.If comprising if, said The special statement being incorporated by reference into included in bright book does not weaken the general statement being incorporated by reference into any way.This Reference in text to bibliography is not intended to recognize：The bibliography is the related prior art, it is not formed on these yet Interior any of perhaps date of publication or file recognizes.With regard to the bibliography provide statement term definition with this For the definition conflict provided in the specification of invention, the definition provided in the description of the invention should be used to explain requirement The invention of protection.

Claims (24)

1. a kind of method for being used to treat the cancer in people patient, the described method includes apply to include anti-IL-10 to the individual The therapeutic alliance of antibody or its antigen-binding fragment and TLR9 activators, wherein the TLR9 activators are CpG-C type few nucleosides Acid.

2. according to the method described in claim 1, wherein described anti-IL-10 antibody be monoclonal antibody, it is humanized antibody, embedding Close antibody or human antibody.

3. according to the method described in claim 1, wherein described anti-IL-10 antibody or its antigen-binding fragment include：(a) SEQ ID NO:5th, 6 and 7 light chain CDR and SEQ ID NO:8th, 9 and 10 heavy chain CDR.

4. according to the method described in claim 1, wherein described anti-IL-10 antibody or its antigen-binding fragment include SEQ ID NO:11 and SEQ ID NO:12 heavy chain and light chain variable region.

5. according to the method described in claim 1, wherein described anti-IL-10 antibody or its antigen-binding fragment are anti-IL-10 Hum 12G8 or its antigen-binding fragment or anti-IL-10 hum 12G8 variations or its antigen-binding fragment.

6. according to the method described in claim 1, wherein described anti-IL-10 antibody is the anti-IL-10 comprising heavy chain and light chain Monoclonal antibody, and wherein described heavy chain includes SEQ ID NO:1, and the light chain includes SEQ ID NO:2.

7. according to the method described in any one of claim 1-6, wherein the CpG-C types oligonucleotides is by following sequence group Into：(a) 5’-N_x(TCG(N_q))_yN_w(X₁X₂CGX₂’X₁’(CG)_p)_z,N_v(SEQ ID NO:13), wherein N is nucleosides, x=0, 1st, 2 or 3, y=1,2,3 or 4, w=0,1 or 2, p=0 or 1, q=0,1 or 2, v=0-89 and z=1-20, X₁And X₁' be From complementary nucleosides, and X₂And X₂' it is from complementary nucleosides；The palindromic sequence of at least eight bases longs, wherein described time (b) Literary sequence includes (X₁X₂CGX₂’X₁’(CG)_p)_zFirst (X of sequence₁X₂CGX₂’X₁'), wherein the oligonucleotides is 12-100 A bases longs.

8. according to the method described in claim 7, wherein x=0, y=1, w=0, p=0 or 1, q=0,1 or 2, v=0-20 And z=1,2,3 or 4.

9. according to the method described in any one of claim 1-6, wherein the CpG-C types oligonucleotides is by TCGN_q (X₁X₂CGX₂'X₁'CG)_zN_v(SEQ ID NO:14) form, wherein N is nucleosides, q=0,1,2,3 or 4, v=0-20, z=1- 4, X₁And X₁' it is from complementary nucleosides, X₂And X₂' it is certainly complementary nucleosides, and wherein described oligonucleotides is at least 12 bases Length.

10. according to the method described in any one of claim 1-6, wherein the CpG-C types oligonucleotides by 5 '- TCGN_qTTCGAACGTTCGAACGTTN_s-3’(SEQ ID NO:15) forming, wherein N is nucleosides, q=0,1,2,3 or 4, s= 0-20, and wherein described oligonucleotides is at least 12 bases longs.

11. according to the method described in any one of claim 1-6, wherein the CpG-C types oligonucleotides have by 5 '- TCGAACGTTCGAACGTTCGAACGTTCGAAT-3’(SEQ ID NO:20) sequence of composition.

12. according to the method described in any one of claim 1-6, wherein the CpG-C types oligonucleotides have by 5 '- TCGTTCGAACGTTCGAACGTTCGAA-3’(SEQ ID NO:17) sequence of composition.

13. according to the method described in any one of claim 1-6, wherein the CpG-C types oligonucleotides is SEQ ID NO:The sodium salt of 17 sequence, and the oligonucleotides is the oligodeoxynucleotide for having phosphorothioate backbone.

14. a kind of method for being used to treat the individual human for being diagnosed to be cancer, the described method includes to described individual with 1-16 weekly Apply SEQ ID NO to the dosage intra-tumor of mg:20 CpG-C types oligonucleotides and the dosage with 1 1-10 mg/kg every 3 weeks Anti- IL-10 hum 12G8 intravenously are applied, are preferably applied with the dosage intra-tumor of 1,2,4,8 or 16 mg weekly SEQ ID NO:20 CpG-C types oligonucleotides and with the dosage of 11,3 or 10 mg/kg every 3 weeks intravenously apply it is anti- IL-10 hum 12G8。

15. a kind of method for being used to treat the individual human for being diagnosed to be cancer, the described method includes to described individual with 1-16 weekly Apply SEQ ID NO to the dosage intra-tumor of mg:20 CpG-C types oligonucleotides 4 weeks, then every 3 weeks 1 time, and with every 3 weeks 1 The dosage of secondary 1-10 mg/kg intravenously applies anti-IL-10 hum 12G8.

16. a kind of method for being used to treat the individual human for being diagnosed to be cancer, the described method includes to the individual the 1st, 8,15, Apply SEQ ID NO to 22 days dosage intra-tumors with 1.0 or 4.0 mg:20 CpG-C type oligonucleotides, then every 3 weeks 1 It is secondary, and anti-IL-10 hum are applied with the dosage of 1 70 mg, 210 mg or 700 mg every 3 weeks intravenously on day 1 12G8。

17. according to the method described in any one of claim 1-15, wherein the cancer is selected from melanoma, neck squamous cell Cancer, breast cancer and non-Hodgkin lymphoma.

18. according to the method described in any one of claim 1-16, wherein the cancer is selected from melanoma, head and neck cancer, breast Gland cancer and B- cell lymphomas.

19. according to the method described in any one of claim 1-15, wherein the cancer is selected from metastatic or can not cut Melanoma, late period neck squamous cell carcinoma, the breast cancer of corium transfer and the painless non-Hodgkin lymphoma removed.

20. according to the method described in any one of claim 1-15, wherein the cancer is selected from clear-cell carcinoma, non-small cell Lung cancer, carcinoma of urinary bladder and colorectal cancer.

21. according to the method described in any one of claim 1-6,14-15,17 and 19-20, wherein the CpG-C types are few Nucleotide is SEQ ID NO:The sodium salt of 20 sequence, and the oligonucleotides is the few deoxidation for having phosphorothioate backbone Nucleotide.

22. according to the method described in any one of claim 16 and 18, wherein the CpG-C types oligonucleotides is SEQ ID NO:The sodium salt of 20 sequence, and the oligonucleotides is the oligodeoxynucleotide for having phosphorothioate backbone.

23. according to the method described in any one of claim 1-6,14-15,17 and 19-20, wherein the CpG-C types are few Nucleotide sequence is SEQ ID NO:20, and the oligonucleotides is the oligodeoxynucleotide for having phosphorothioate backbone.

24. according to the method described in any one of claim 16 and 18, wherein the CpG-C types oligonucleotide sequence is SEQ ID NO:20, and the oligonucleotides is the oligodeoxynucleotide for having phosphorothioate backbone.